SUMMARY: The diffusion of streptomycin in agar gel seeded with staphylococci as in assay methods, results in a clearly defined zone of inhibition. The time required for this definition to develop is about Z& hr. at 37", but varies greatly with tem- perature and is equal to the lag period plus approximately 4 times the generation time. After 4-5 generations the young colonies become much less susceptible and grow unimpeded by the rapidly increasing concentration of the antibiotic reaching them. Incubation at temperatures below 87O, although without significant effect on the minimum inhibitory concentration, results in larger inhibition zones because of increased generation times. In assays and sensitivity tests by diffusion methods, errors will occur if organisms under comparison do not multiply at the same rate. When an antiseptic diffuses into nutrient agar seeded with a sensitive organism (as in the standard diffusion assay methods) there is a race between the drug diffusing into the medium, and the growth of the colonies. At first sight it might seem that the edge of the zone of inhibition occurs at a point where colonies have grown to macroscopically visible size or to full size before the minimum inhibitory concentration of the antiseptic reaches them. This explanation, however, does not account for the sharpness of the edge of the inhibited zone seen with some antibiotics. Experiments with streptomycin indicate that when the young colonies have grown for a certain critical, and quite short, time, they become much less susceptible to the action of strepto- mycin, and that they continue to grow to full and even to enhanced size in spite of the presence around them of much more than the minimum inhibitory Concentration. Thus the edge of the zone is defined before the colonies become visible to the naked eye. The experiments with streptomycin using the Mayo strain of staphylococcus were concerned in the first place with the effect of temperature on the size of the inhibited zone. The formula which we used was suggested by Cooper & Woodman (1946). This formula relates the distance travelled by the minimum inhibitory concentration of antiseptic in any particular time to the concentra- tion of the antiseptic placed in the cup or on top of the column of agar. It rests on theoretical principles which apply to the tube assay method for strepto- mycin (described by Mitchison & Spicer, 1940) where the agar seeded with the organism is placed in narrow tubes, and the streptomycin is placed on top of the agar column. After incubation the length of the resulting zone of inhibition is measured with a microscope vernier. In the equation of Cooper & Woodman ( 1946) ' 22 = 4( 2-30 DT) (log m, - log m') ;