Utilizing column immunoabsorbent anti-Fab chromatography and rosette depletion techniques three distinct subsets of human lymphocytes were isolated: (1) T cells which are surface Ig− and E rosette positive, (2) B cells which are Ig+, E rosette negative, and (3) a Null cell population which is both Ig− and E rosette negative. All three populations were analyzed with respect to a variety of assays of cell mediated immunity. The Ig−, E rosette negative (Null cell) population exhibited close functional similarities to B lymphocytes. Both responded poorly to plant lectins, did not proliferate in response to soluble or cell surface antigens, and were not active in cell mediated lympholysis. However, both were efficient effector cells in antibody dependent cellular cytotoxicity. T lymphocytes, in contrast, were highly active in proliferative responses to plant lectins and antigens, were efficient in cell mediated lympholysis, but were not active effector cells in antibody dependent cellular cytotoxicity. These studies support the view that the human Ig−, E rosette negative cells represent a subset of B lymphocytes.