The ability of recombinant interferons (IFNs) to modulate recombinant interleukin-2 (rIL-2) augmentation of natural killer (NK)-cell activity and to modulate the generation of activated killer (AK) cells was examined. Incubation of murine spleen cells for 18 hr with either human rIL-2 or a human hybrid recombinant IFN alpha, rHuIFN-alpha A/D, which is active on murine cells, resulted in a dose-dependent increase in NK activity; however, recombinant murine IFN gamma, rMuIFN-gamma, had little activity. A more than additive augmentation of cytotoxicity was obtained when spleen cells were incubated with the combination of rIL-2 and rHuIFN-alpha A/D. Incubation of murine spleen cells with rIL-2 for 3 days resulted in a dose-dependent induction of AK cells which were cytotoxic to an NK-resistant tumor target cell. In contrast to the results observed on NK activity, incubation of spleen cells with rHuIFN-alpha A/D and rIL-2 inhibited AK-cell activity. Partially purified murine IFN-alpha had inhibitory activity comparable to that of rHuIFN-alpha A/D. The addition of rHuIFN-alpha A/D at the initiation of culture of spleen cells with rIL-2 (day 0) resulted in maximal inhibition of cytotoxicity; inhibition was reduced or absent if rHuIFN-alpha A/D was added on day 1 or 2 of culture. The proliferation of spleen cells incubated with rIL-2 was also inhibited by rHuIFN-alpha A/D. Addition of rMuIFN-gamma to spleen-cells and rIL-2 increased the cytolytic activity of AK cells and did not inhibit rIL-2-induced proliferation of spleen cells. Similar data were also obtained with human peripheral blood lymphocytes and recombinant cytokines. Incubation of human peripheral blood lymphocytes with rIL-2 and recombinant human IFN-alpha A (rHuIFN-alpha A) or recombinant human IFN-gamma (rHuIFN-gamma) resulted in a more than additive increase in NK activity. Human AK-cell cytotoxicity was inhibited by rHuIFN-alpha A but enhanced by rHuIFN-gamma. Thus recombinant IFNs have differential effects on rIL-2-induced cytotoxic cells, resulting in augmentation or inhibition of activity, which is dependent on both the type of IFN and the cytotoxic activity examined. These results may have important implications for the potential therapeutic use of combinations of these cytokines.