In the present study, we have analyzed the effects of anti-T8 and anti-T3 monoclonal antibodies (mAb) on the generation of human allospecific cytolytic T lymphocytes (CTL). A sensitive limiting dilution microculture system, in which graded numbers of responder T cells were added to 10(5) irradiated allogeneic spleen cells and saturating amounts of exogeneous interleukin 2, has been employed to provide minimal estimates of the frequencies of alloreactive CTL precursors (CTL-P) present in T lymphocyte populations freshly isolated from peripheral blood. Addition of anti-T8 or anti-T3 mAb at the onset of micro mixed lymphocyte culture containing peripheral blood T lymphocytes as responding cells caused a 65-80% reduction in the frequency of specific CTL-P which proliferate under these conditions. Moreover, in most instances (greater than 80%) the cytolytic activity of those CTL-P which underwent clonal expansion in the presence of either anti-T8 or anti-T3 mAb was found to be resistant to inhibition by the corresponding mAb added during the cytolytic assay. In addition, a great degree of overlap existed between microcultures resistant to anti-T3 and those resistant to anti-T8 mAb. In contrast, neither antibody had any inhibitory effect when added later to the cultures, presumably because at this stage CTL-P had been already triggered by alloantigens. Taken together our data indicate that the heterogeneity in the susceptibility to inhibition by anti-T3 or anti-T8 mAb, previously observed at the level of CTL clones, is predetermined already at the level of peripheral blood CTL-P.