Mutations in ras genes which result in transforming gene products carrying amino acid substitutions in position 12, 13 or 61 are common in human cancer. Peptides encompassing these mutations in ras are shown to be immunogenic in both mice and humans. The potential usefulness of such peptides in cancer therapy, depends on their ability to bind to HLA molecules. We therefore stimulated T cells from healthy donors with mutated ras-derived peptides. By repeated in vitro stimulation of peripheral blood mononuclear cells, several T cells clones could be generated which recognized a p21 ras derived peptide carrying a position 12 Gly-->Arg substitution. This peptide (1-25,12 Arg) could be specifically recognized by T cells restricted by either HLA-DQ7 or -DP3. Previously, we showed that this peptide is also recognized by a T cell clone restricted by HLA-DR2. The core region of the peptide was determined to span positions 9-16 for all three HLA restriction elements, and accordingly contains the mutational hot spots in position 12 and 13. The observation that the mutant 1-25,12 Arg ras-derived peptide may contain a promiscuous epitope encompassing the Gly-->Arg mutation in position 12 indicates that lack of peptide presentation by given HLA molecules may not be a major constraint in responsiveness against ras mutations.