A cloned subline of the human monocytic leukemia cell line, THP-1, was induced to produce high levels of cytotoxic activity following an 18-hour phorbol myristate acetate (CAS: 16561-29-8) stimulation in vitro. This activity, termed monocyte cell line cytotoxin(s) (MCCT), was tested in vitro on different human continuous cell lines (Chang, ESH-7, GM3104A, HeLa, HT-1080, K562, Mel, T-24) and on primary human fibroblasts (GM3468, Manz). The continuous cell lines exhibited a spectrum of sensitivity to MCCT-containing supernatants whereas the primary fibroblasts were resistant to lysis. Enzymatic degradation and heat denaturation studies indicate that MCCT is a protein. Its bioactivity can be resolved into three lytic peaks after molecular sieving on Ultrogel AcA 44. The major peak, designated alpha MCCT, eluted with a molecular weight of 100,000-140,000 daltons. A minor peak, beta MCCT, was seen at 60,000-80,000 daltons, and a third, unstable minor peak, gamma MCCT, eluted at less than 10,000 daltons. The alpha-lytic peak was examined further and was found to migrate as a single peak in 7% native polyacrylamide gel electrolysis tube gels with an rf of 0.25-0.30. None of the MCCT forms were immunologically cross-reactive with human alpha-lymphotoxin. Various protease inhibitors known to inhibit monokine- and macrophage-mediated direct cell lysis in vitro were tested for their inhibitory effects on alpha MCCT activity. The irreversible binding inhibitor N alpha-p-tosyl-L-lysyl chloromethyl ketone inhibited the biologic activity of alpha MCCT. The reversible binding inhibitors N alpha-p-tosyl-L-arginine methyl ester and soybean trypsin inhibitor were able to block in vitro lytic activity when added to alpha MCCT in the presence of the target cell. In contrast, the inhibitors phenylmethylsulfonyl fluoride, L-1-tosylamide, 2-phenylethyl chloromethyl ketone, and N alpha-acetyl-L-lysine methyl ester were not effective in blocking cytolysis. Finally, the hydrogen peroxide scavenger catalase inhibited alpha MCCT lytic activity in vitro; however, the hypochlorous acid scavengers alanine, serine, and valine were without effect.