Patients with melanoma metastatic to distant sites or at high risk for recurrent melanoma have been treated with a polyvalent melanoma cell vaccine (MCV) in phase II protocols. We assessed in vivo and in vitro cell-mediated responses to MCV in 163 patients who had undergone surgical resection of American Joint Committee on Cancer stage III melanoma. During the first 4 months of vaccine immunotherapy, 135 patients (83%) responded by developing a positive delayed-type hypersensitivity reaction > or = 6 mm to MCV. In a mixed lymphocyte tumor cell reaction using peripheral blood lymphocytes, 35 of 42 patients (83%) showed a recall proliferative response to one or more of the three cell lines of MCV. There was a significant correlation between delayed-type hypersensitivity reaction and mixed lymphocyte tumor cell reaction (P = 0.013). After 4 months of MCV therapy, 8 of 11 patients had an increased mixed lymphocyte tumor cell reaction to autologous melanoma cells. During the first 4 months of vaccine therapy, 16 of 33 patients developed more than a 50% increase in cytotoxic T-cell activity against one of the cell lines of MCV. Overall survival was significantly prolonged in patients with a positive delayed-type hypersensitivity reaction (P = 0.0054) and/or increased cytotoxic T-cell activity (P = 0.02). These findings suggest that MCV induces specific T-cell responses which are correlated with clinical course; the data also suggest that some of these responses are directed against autologous melanomas and may play a major role in controlling the progression of melanoma.