To probe the mechanism of suppressor T cell generation in man, we have carried out mixed leukocyte reactions (MLR) in the presence of cyclosporin (CsA), a fungal metabolite which prevents the generation of cytotoxic lymphocytes while permitting activation of suppressor cells. After a 12-d MLR in the presence of 1 microgram/ml CsA, T cells were fractionated into subsets with monoclonal antibodies, and each subset was tested for the ability to inhibit a second fresh MLR that is devoid of CsA. The results show that Leu 2+ T cells derived from the first culture suppress the second MLR in an HLA-DR antigen-specific manner and in the absence of detectable lysis of stimulator cells. However, Leu 2+ cells do not develop into suppressor cells unless acted upon by alloantigen-primed Leu 3+ inducer cells. Furthermore, only those Leu 3+ cells that also express the Leu 8 marker (Leu 3+, 8+) are capable of inducing suppressor cells. Thus, antigen-specific feedback inhibition of an immune response in man results from an ordered series of interactions between T cells of distinct phenotype.