Subpopulations of human peripheral blood leukocytes were isolated by rosette formation and tested for functional activity. E -rosette-forming cells (E-RFC) and EAC-RFC were separated from non-rosetting cells by sedimentation on Ficoll-Hypaque gradients. The efficiency of the method and the purity of the resulting subpopulations were high. E-RFC responded to PHA Con A, allogeneic leukocytes, and PPD, with higher levels of proliferative reactivity than the unseparated lymphocytes while E-RFC depleted, EAC-RFC, and null cells showed only low levels of reactivity. Reactivity to PWM and tetanus toxoid was also restricted to the E-RFC subpopulation, but was lower than that of unseparated cells. A staphylococcal antigen preparation triggered lymphoproliferative reactivity in the E-RFC, E-RFC deleted, EAC-RFC, and the null cell subpopulations. 51Cr release lymphocyte cytotoxicity against a human lymphoblast target cell line was found in the E-RFC and null cell fractions but was not observed with the EAC-RFC subpopulation.