Studies have been made on the ultracentrifugal fractionation of chorio-allantoic fluid from embryos infected with the PR8 strain of influenza A virus. By means of alternate low- and high-speed ultracentrifugal cycles, a particulate component of 77.6 mμ diameter and a sedimentation-constant of S20° = 724 × 10-13 has been isolated. An identical component has been obtained by ultracentrifugation of eluate of red blood cells agglutinated by virus-infected chorioallantoic fluid and of virus adsorbed on calcium phosphate. Tests of the various fractions made quantitatively within the limits of the methods by red blood cell agglutination, specific precipitation, complement fixation and infectivity have indicated an association of the virus with these particles. Two papers describing experiments with influenza virus, previously reported briefly (2, 27), were recently published, one by Chambers and Henle (7) and the second by Chambers, Henle, Lauffer and Anderson (8). These authors reported the concentration of a material of 10 to 12.5 mμ particle-diameter which was considered to be the virus of influenza. In addition, a component with a sedimentation-constant of S20° = ca800 ± 100 × 10-13 was observed in sedimentation-velocity studies, which “was shown to consist principally of aggregated particles of the more disperse fraction.” In general, the methods employed in this laboratory were somewhat similar to those used by Chambers and his associates, yet the conclusions reached were in some respect wholly dissimilar. Although a complete explanation of the differences in conclusions is not apparent, certain aspects of the present work will be discussed with consideration of the results of Chambers and his co-workers. The chorio-allantoic fluid used for the present purification-process was not frozen, since freezing might have resulted in loss during initial clarifying low-speed centrifugations. It has been shown that a precipitate is developed in chorio-allantoic fluid on thawing from the frozen state, and this has been made the basis for concentration (4) of the virus. After preliminary experiments, the fluid was dialyzed immediately on removal from the embryos, since this procedure eliminated the urates which otherwise sedimented in the first ultracentrifugal cycle. The use of calcium chloride was based on previous studies on equine encephalomyelitis virus (19), wherein it was found difficult to demonstrate in the electron microscope virus particles in the numbers expected on the basis of known concentration in the preparations. While studying the influence of various salts on the Eastern strain virus, preparations were made by dilution with calcium chloride solution. Films of such preparations showed a difference from those diluted with water or saline in the even dispersion of the approximate expected number of particles in electron micrographs. The contrast of the particle-images was greater and salt crystals were infrequent. It was thought that the phenomenon might be related to the hygroscopic properties of the salt in preventing violent drying currents and consequent removal of virus from the field with salt crystallization. Another possible explanation for this effect is the known (28) dispersing action of calcium chloride, which may have acted to maintain uniform suspension and spreading of the virus on the film. The same phenomenon was observed with the concentrates associated with influenza virus, for, while the large particles were routinely demonstrable in the presence of calcium chloride of 0.023 M concentration, satisfactory demonstration of this component in water preparations was difficult. The inclusion of calcium chloride in the purification-process was based on the previous observations and on additional findings in the present work. Ringer solution was first used as the solvent, but it was found that when ultracentrifugal concentrates were taken up in Ringer solution alone, a large part of the viral activity was lost in the low-speed cycle in the ultracentrifuge or the angle centrifuge. In the hope of minimizing this loss, calcium chloride in 0.023 M additional concentration was included in the solvent for the entire procedure. Under these conditions, the viral activity lost in low-speed centrifugation was markedly reduced, indicating more complete re-dispersion of the particles packed by ultracentrifugation at 27,000 g. Attempts were made to purify the concentrate, using 0.1 M salt solution. At this concentration of salt, the particles were partially insoluble and nearly all of the viral activity was lost in the low-speed runs. Control studies showed that calcium chloride, as used, did not affect the specific relations in the biological tests employed. The experiment combining elution from red blood cells and ultracentrifugal concentration was carried out on the basis of possible selective adsorption (15), in the hope of obtaining thereby the specific viral particles freed from extraneous material present in chorio-allantoic fluid. The material removed by the agglutinated red blood cells, and subsequently concentrated by ultracentrifugation, was predominantly of about 80 mμ particle-diameter, as shown by the electron microscope (figs. 3 and 4). Small particles were plentiful in the supernate of the chorio-allantoic fluid after red blood cell adsorption, but no evidence of them was seen in the sedimentation-velocity diagrams of the concentrated eluate (fig. 6) and relatively few were seen in the electron micrographs (figs. 3 and 4). The small particles were obtained in profusion from the adsorbed supernatant fluid after subjecting it to higher-speed ultracentrifugation (67,000 g for 2 hours). Little activity was associated with such material, and that seen could be accounted for on the basis of a small amount of the larger component seen in the electron micrograph of figure 8. In addition,...