In prior studies a heteroantiserum to a surface membrane component termed T(H2) was used to define two subsets of human T cells (T(H2) (+) and T(H2) (-)), which were found to express distinct sets of activities in vitro. In 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 alphaLeu-2a and alphaLeu-2b, which seem to define the same surface antigen identified by the original T(H2) antiserum, reacted with 57-84% of thymocytes and 22-46% of the erythrocyte-rosette-forming cells (ERF-C) in peripheral blood. Two other monoclonal antibodies, termed alphaLeu-3a and alphaLeu-3b, reacted with the same subpopulation of thymocytes (78-89%) and peripheral blood ERF-C (47-78%) but, unlike alphaLeu-2a and alphaLeu-2b, did not exhibit cross-blocking; i.e., labeling cells with alphaLeu-3a did not inhibit the subsequent binding of alphaLeu-3b. T cells reactive with alphaLeu-2a were shown to be unreactive with alphaLeu-3a, indicating that two separate subpopulations of T cells, Leu-2 (formerly T(H2) (+)) and Leu-3 (T(H2) (-)) 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-1(+) 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 of complement. These experiments established the following points: (i) alphaLeu-2a abolished the killing by cytotoxic T cells of allogeneic phytohemagglutinin-stimulated blasts, (ii) inhibition of killing by alphaLeu-2b was markedly less than inhibition by alphaLeu-2a, and (iii) other antibodies, including alphaLeu-1, alphaLeu-3a, and alphaLeu-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.