Interest in semi-allogeneic vaccines has been increasing, as demonstrated by the publication of successful preclinical and clinical studies by others and us that validate this immunotherapeutic approach to cancer and HIV-infection. We now report that lymphocytes from an HLA-A2+ melanoma patient, stimulated with a GP100-derived epitope and semi-allogeneic hybrids, lysed target cells presenting this peptide more efficiently than lymphocytes stimulated with GP100 peptide alone. Phenotypic analysis with GP100/HLA-A2 tetramer complexes also demonstrated a significant increase in the size of the GP100-specific CD8+ lymphocyte pool when PBMC were stimulated with both peptide and semi-allogeneic hybrids. Analyses of PBMC from other donors further suggest that this stimulatory effect of semi-allogeneic hybrids results from an increase in the HLA-restricted recall response of CD8+ cytotoxic T lymphocytes against melanoma-derived antigens. Likewise, lymphocytes from an HIV-infected patient that had been stimulated with a mixture of HIV-derived peptides and semi-allogeneic hybrids also demonstrated significantly greater antigen-specific cytotoxicity and tetramer reactivity compared with those lymphocytes that had been stimulated with peptides alone. Our approach should provide useful information for the design of future clinical studies treating patients with melanoma or HIV with therapeutic vaccines consisting of a combination of semi-allogeneic hybrids and immunodominant antigenic peptides.