In prior studies a heteroantiserum to a surface membrane component termed TH2 was used to define two subsets ofhuman T cells (TH2 and TH ), which were found to express distinct sets of activities in vitra2In the present studies we prepared monoclonal antibodies to surface determinants that are restricted to T cells belonging to, each of these two subsets. Two antibodies, termed aLeu-2a and aLeu-2b, which seem to define the same surface antigen identified by the original T 2 antiserum reacted with 57-84% of thymocytes and 22-46% oP the erythrocyte-rosette-forming cells (ERF-C) in peripheral blood. Two other monoclonal antibodies, termed aLeu-3a and atLeu-3b, reacted with the same subpopulation of thymocytes (78-89%) and peripheral blood ERF-C (47-78%) but, unlike aLeu-2a and aLeu-2b, did not exhibit cross-blocking; i.e., labeling cells with aLeu-3a did not inhibit the subsequent binding of aLeu-3b. T cells reactive with aLeu-2a were shown to be unreactive with aLeu-3a, indicating that two separate subpopulations of T cells, Leu-2 (formerly TH2 ) and (TH2-) T cells, were thereby defined. These two T cell subsets make up the subpopulation of ERF-C (80-95%) previously defined by a monoclonal antibody to a T cell membrane antigen (Leu-1) that has a thymus-dependent distribution on normal lymphocytes but is expressed by some surface-immunoglobulin-positive (sIg') leukemic lymphocytes. None of the Leu antibodies reported here reacted with sIg', Leu-1I leukemic cells, nor did they react with normal hematopoietic cells or lymphoid cells that had surface markers characteristic of B cells. Studies of the blocking effects of Leu antibodies on killing in cell-mediated lympholysis by effector T cells were carried out in the absence ofcomplement. These experiments established the following points: (i) aLeu-2a abolished the killing by cytotoxic T cells of allogeneic phytohemagglutinin-stimulated blasts, (ii) inhibition of killing by aLeu-2b was markedly less than inhibition by aLeu-2a, and (iii) other antibodies, including aLeu-1, aLeu-3a, and aLeu-3b, had little or no effect on killing in cell-mediated lympholysis. The relevance of these findings to prior studies done in the mouse and -in man are discussed.There is now good evidence in man, as in the mouse, that the thymus gives rise to functionally distinct subpopulations of T cells that can be defined by their unique surface phenotypes of thymus-dependent membrane antigens (1-7). This was demonstrated by the use of heterologous anti-human T cell sera (6, 7) and has been explored more recently by using monoclonal antibodies generated by somatic cell hybridization techniques (8,9). These studies have shown that a thymus-dependent antigen, termed TH2, is expressed on 30-40% of circulating T cells, and that TH2+ T cells mediate suppressor effects in vitro and are also responsible for most of the killing in cell-mediated lympholysis (CML) (7, 9). In contrast, TH2-T cells were shown to effect markedly less cytotoxic activity in CML but were found to amplify the functions...