IFN-alpha2b has been used to treat patients with malignant melanoma who are at high risk for recurrence after surgical resection. However, its exact mechanism of action is unknown. I hypothesized that IFN-alpha exerts a direct effect on the melanoma cell via the activation of signal transducer and activator of transcription (STAT) proteins. Cell lysates from melanoma cell lines and patient tumor samples stimulated with IFN-alpha were incubated with radiolabeled oligonucleotides representing the high affinity sis-inducible element of c-fos and the IFN stimulated response element and then analyzed for STAT activation using the electrophoretic mobility shift assay. Melanoma cell lines showed no evidence for constitutive STAT activation in the absence of cytokine stimulation but exhibited rapid activation of STAT1 and STAT2 once treated with IFN-alpha. A distinct dose-response curve was noted with maximal STAT activation occurring at approximately 10(5) units/ml IFN-alpha. Genistein, a protein tyrosine kinase inhibitor, completely suppressed IFN-alpha-induced STAT activation. Malignant melanoma tumors obtained from 17 patients exhibited dose-dependent activation of STAT1 and STAT2 in response to treatment with IFN-alpha. Pretreatment of patient melanoma tumor cells with IFN-gamma resulted in a 4 log-fold decrease in the IFN-alpha concentration required for STAT activation and promoted the increased expression of STAT1, STAT2, and p48. In summary, IFN-alpha consistently activated STAT1 and STAT2 proteins in melanoma cell lines and in melanoma tumors obtained directly from patients. This signaling pathway was dramatically sensitized to IFN-alpha by pretreatment of melanoma cells with low concentrations of IFN-gamma. These results provide molecular evidence to support the hypothesis that the clinical response to IFN-alpha may be mediated, in part, by a direct effect on the melanoma cell. These results also suggest a potential role for IFN-gamma in the treatment of malignant melanoma.