In this study we compared several protocols for in vitro induction of cytotoxic T lymphocytes (CTL) from naïve HLA-A*0201(+) peripheral blood mononuclear cells (PBMC) against allogeneic melanoma cells. As immunization material we compared: (1) the lysate of apoptotic or live melanoma tumor cells [MSM-M14 (M14) and MSM-M3 (M3)]; and (2) total RNA extracted from the same melanoma cell lines, either unconjugated or conjugated with a charged carrier (DMRIE-C). Overall killing activity was very similar in CTL induced by tumor lysate or RNA. CTL induced by both methods preferentially killed an HLA class I-matched M14 melanoma cell line rather than HLA class I-unmatched M3 cells. Cytotoxicity could be partially blocked by anti-HLA class I antibodies. There were no significant differences in cytotoxicity and in other clonal characteristics in CTL lines induced by a lysate of apoptotic bodies as compared to lines induced by lysate of viable cells. However, CTL induced by DMRIE-C-bound total RNA demonstrated superior cytotoxicity when compared with CTL induced by unconjugated total RNA. Polyclonal CTL induced by tumor lysate contained a substantial percentage of tyrosinase(368-376 370N) tetramer-positive cells and demonstrated specific killing activity against tyrosinase(368-376 370N) peptide-labeled T2 cells, comparable to cytotoxicity of the CTL developed against this peptide alone. In contrast, there were no detectable tyrosinase(368-376 370N)-tetramer positive cells and no specific anti-tyrosinase peptide(368-376 370N) response in polyclonal CTL induced by immunization with tumor RNA. These data demonstrate that both total tumor RNA and tumor lysate are effective for inducing of cytotoxic anti-melanoma CTL, but tyrosinase(368-376 370N) specific cells were detected only in lysate-induced CTL cultures. This suggests that nature of the antigens present in tumor lysate might be different from those in tumor RNA.