Activated killer (AK) cells cytotoxic for freshly prepared autologous melanoma cells were generated in vitro when recombinant interleukin 2 (rIL2) was incubated with peripheral blood mononuclear cells from 11 of 12 patients with metastatic melanoma. The cytotoxic response first appeared significant after 3 days of culture with rIL2; it peaked after 6 days and then declined after 9 days of incubation. Peripheral blood mononuclear cells cultured with medium alone (control) or with recombinant gamma-interferon alone (200 units/ml) failed to acquire AK cytotoxicity at any time in any of the 12 patients. Autologous tumor cells also could not induce AK activity in mixed-lymphocyte tumor cell cultures. The level of AK activity generated was significantly reduced (P less than 0.001) when 10% autologous human serum was used instead of 10% fetal calf serum in the rIL2 cultures. This suppression was specific for the inductive phase of AK activity, because autologous human serum could not suppress the cytotoxic capability of AK cells once they were activated by rIL2. The serum suppressive effect on the induction of AK cytotoxicity could be overcome by increasing the dose of rIL2 or by adding recombinant gamma-interferon to the cultures. Lymphocytes from melanoma patients thus have the latent ability to kill autologous melanoma tumor cells. However, this cytotoxic capability requires an interleukin 2-induced differentiation step that could be amplified by gamma-interferon and inhibited by a serum suppressive factor.