The role of E rosette-forming cells in leukocyte migration inhibition was investigated with human buffy coat cells fractionated by combined E rosette formation and Ficoll-Hypaque (FH) density gradient centrifugation. The migration of unfractionated dextran-sedimented leukocytes and FH-separated mononuclear cells (MN) were equally inhibited in the presence of Escherichia coli somatic antigen (E. coli), purified protein derivative of tuberculin (PPD) and influenza virus antigen (Flu). Polymorphonuclear leukocytes (PMN) separated from whole leukocytes by one FH density gradient fractionation contained greater than 1.0% E rosette-forming cells and were inhibited by E. coli and PPD. PMN submitted to three transits of FH (DF III) contained less than or equal to 0.02% E rosette forming cells and were unaffected by each of the three antigens. The migration of mixtures of 10% MN and 90DF III cells were inhibited in the presence of specific antigen, while MN depleted of E rosette-forming cells (less than or equal to 0.5%) pooled with equal numbers of DF III cells did not respond to any antigen by migration inhibition. These results show that PMN purified by three transits of FH are unable to respond to specific antigen by inhibition of migration from microcapillary tubes, and specific antigen-induced inhibition of migration of peripheral blood leukocytes does not occur in the absence of D rosette-forming cells in normal subjects.