Human alloreactive cytotoxic T lymphocytes (CTLs) were generated in culture by stimulating peripheral blood lymphocytes against lymphocytes of unrelated donors. OKT3 monoclonal antibody, which reacts with all human peripheral T cells, inhibited more than 50% of the lysis of the allogeneic cells by the CTLs in the four-hour cytotoxicity assay at a concentration of 1 microgram ml-1. OKT4, which reacts with inducer/helper T cells, did not demonstrate this effect. OKT5 and OKT8, both of which react with cytotoxic/suppressor T cells, had a small but distinct inhibitory effect (about 20-30% maximally). OKT3 antibody is also known to induce mitogenesis of peripheral T cells, but this mitogenic effect is probably of itself not the cause of the inhibitory effect of OKT3 on CTLs. Human plasma abolished OKT3-induced mitogenesis of T cells but not OKT3-mediated inhibition of CTL activity. In addition, there exist conditions that allow both full mitogenesis and intact CTL activity, suggesting CTLs can maintain cytolytic activity under mitogenic conditions. The generation of CTLs in the 6-day mixed lymphocyte culture was also inhibited by OKT3. Several possible mechanisms for this effect are discussed.