We have investigated the influence of mutations in the Ras 61 codon on the immunogenicity of synthetic peptides and H-Ras p21 proteins. H-2k mice produced Th responses when immunized with mutated peptides in which the Gln at position 61 in the wild type sequence was replaced by Leu (L) or His (H). T cell hybridomas specific for the 61L and 61H peptides were then produced. The responses of both were I-Ak restricted. Competition experiments indicated that the wild type peptide did not bind to the I-Ak molecule whereas the two mutations generated a site on the peptides that was agretopic for the I-Ak molecule. Nevertheless the recognition of the corresponding Ras proteins was highly dependent upon the nature of the substitution. The H-Ras p21 protein with the 61L mutation (61L) was processed by syngeneic splenocytes and the epitope dependent on 61L was recognized as efficiently as the corresponding peptide by the T cell hybridoma specific for 61L. In contrast, the processing of H-Ras p21 with the 61H mutation (61H) was probably inefficient in producing the epitope recognized by the hybridoma specific for 61H. Furthermore, immunization studies with the two mutated H-Ras p21 proteins suggest that only the 61L substitution can be exploited for immunotherapy. Thus this work demonstrates that any peptide immunotherapy must be undertaken with the reservation that not all oncogenic mutations at codon 61 will be amenable to immune therapy.