Mononuclear leukocytes were separated from whole blood by ficoll-hypaque flotation, and by elutriation (counterflow centrifugation). Lymphocytes isolated from 6 control subjects by elutriation showed a 30% greater response to stimulation with phytohemagglutinin and 130% greater response to streptokinase-streptodornase stimulation than did autologous lymphocytes obtained by ficoll-hypaque separation. Cell yields of major mononuclear cell subpopulations and cell viability were comparable after separation of leukocytes by both techniques. These results indicate that ficoll-hypaque flotation may diminish lymphocyte responses, and that elutriation offers a useful alternative to ficoll-hypaque separation. In addition, elutriation may be the preferable method for evaluation of lymphocytes from patients with suspected immunologic dysfunction and may be valuable in the isolation of mononuclear cells from infiltrated skin lesions.