Three categories of tumor promoters and chemically related but inactive substances were tested for their effect on the cytotoxic activity of human blood lymphocytes against K562 and Daudi targets. Lymphocytes incubated overnight in the presence of phorbol esters 12-O-tetradecanoylphorbol-13-acetate and phorbol-12,13-dibutyrate [P(Bu)2] had enhanced function. Incubation with 4-alpha-phorbol-12,13-didecanoate was without effect. Enhancing activity was also exerted by the indole alkaloids, teleocidin and lyngbyatoxin A, and the polyacetates, aplysiatoxin and debromoaplysiatoxin, but not by dihydroteleocidin. Only the tumor-promoting compounds activated the cytotoxic potential. The substances acted in a dose-dependent manner with optimal activity at characteristic concentrations. Overnight incubation of lymphocytes at 4 degrees did not change their spontaneous cytotoxicity but abolished the enhancing effect of P(Bu)2. Thus, P(Bu)2-induced activation occurred only on metabolically active cells. The activation did not require DNA synthesis. Similar to controls, the P(Bu)2-treated cells required divalent cations and an intact cytoskeleton in order to perform their lytic function. Experiments with the various metabolic inhibitors indicate that phorbol ester treatment does not induce an alternative cytotoxic mechanism since, as with untreated lymphocytes, P(Bu)2-activated cells require contact with the target and intact secretory functions. The enhanced cytolytic potential was not due to induction of alpha-interferon (IFN-alpha) production, as shown by the fact that the effect was not abolished by addition of anti-IFN antibodies during the P(Bu)2 treatment of lymphocytes or during the cytotoxic assay. However, the presence of antiserum against IFN reduced the cytotoxic potential of control cells, suggesting that endogenous IFN production contributes to the maintenance of lytic function in cultured cells. If this mechanism is counteracted by addition of anti-IFN serum, the phorbol esters can provide an alternative activation signal. When P(Bu)2-activated lymphocytes were subsequently treated with IFN-alpha or IFN-gamma, their lytic capacity was further increased. These results indicate that P(Bu)2 and IFN activate cytotoxic potential through different pathways.