Supernatants of hybridomas from fused spleen cells of mice immunized with either a T4+ or a T8+ CTL clone, were screened for their ability to inhibit the cytotoxic activity of the T4+ and the T8+ CTL clone. Eight monoclonal antibodies (MAbs) with blocking activity were obtained and a preliminary characterization of these antibodies was carried out. The MAbs SPV-L1 and SPV-L5 were found to react with thymocytes and all peripheral blood leukocytes. SPV-L1 and SPV-L5 precipitated a molecular complex consisting of two noncovalently bound chains of molecular weights of 95 and 160 kD indicating that they recognize the human equivalents of the recently described leukocyte function associated (LFA-1) antigens. The SPV-L1 and SPV-L5 antibodies inhibited the reactivity of six CTL clones tested in this study. In addition, SPV-L1 and L5 blocked the lectin dependent cellular cytotoxicity mediated by these CTL clones. Natural killer cell activity mediated by fresh peripheral blood lymphocytes and activated killer cell activity generated in MLC both measured against K562 was also inhibited by SPV-L1 and SPV-L5. Two other antibodies, SPV-L3 and SPV-L4 blocked strongly the cytotoxic activity of two T4+ CTL clones, whereas the reactivity of the three T8+ CTL clones and one T4+ CTL clone was not or only moderately inhibited. These antibodies recognized an Ia antigen as judged from tissue distribution and immunoprecipitation studies. However, these latter studies also suggest that SPV-L3 and SPV-L4 recognize HLA-DC rather than HLA-DR antigens. Finally, three antibodies (SPV-T3a, SPV-T3b, SPV-T3c) were obtained directed against the T3 molecular complex and one MAb (SPV-T8) was found to react with T8. The anti-T3 reagents were shown to be mitogenic for peripheral blood lymphocytes, with the exception of SPV-T3a. The results presented here indicate that the antigens recognized by SPV-L1 and SPV-L5 are involved in various cytotoxic reactions. In contrast, SPV-L3, SPV-T3 and SPV-T8 only seem to play a role in antigen specific cytotoxic reactions.