We have expanded our characterization of the response of autologous lymphocytes to cultured melanoma and our exploration of associated humoral immune phenomena. Our data on cellular immunity continue to support the hypothesis that biologically early melanoma provokes autologous lymphocyte stimulation and that metastatic disease does not. Four additional avenues have been explored. First, we have begun studies to characterize the phenotype of the lymphoid population responding to autologous melanoma. We employ commercially available monoclonal antibodies to label autologous lymphocytes of unstimulated control cultures and cocultures stimulated by autologous melanoma, autologous B cells transformed by EBV and the allogeneic B-lymphoblast line, Raji. Second, we have established a reproducible assay for cytotoxicity. Using this assay we have demonstrated that cytotoxicity is generated in autologous cocultures. Thus, lymphocyte proliferation not only expands the population of cytotoxic cells but is accompanied by a functional consequence of lysis of autologous targets. Third, we are exploring an assay for lymphocyte proliferation that requires far fewer stimulating cells than that employing 3H-TdR. This exploits the dramatic expansion of the T9 phenotype associated with lymphocyte stimulation. Finally, we have addressed several problems in using cultured lines, such as contamination of cell lines by other cell lines. With respect to humoral immunity, we have applied the solid phase RIA to a number of autologous pairings of melanoma cells and serial patient sera. To date, we have found significant binding above base line only occasionally.