Recently, we provided the first direct evidence that the determinant recognized by some alloreactive cytotoxic T lymphocytes (CTL) clones included an endogenous peptide antigen(s), which could be derived by cyanogen bromide cleavage of cytoplasmic proteins. The studies outlined in this report provide three important findings which confirm and extend our previous results. First, the description of peptide-dependent Kb-specific alloreactive CTL clones is now extended to clones isolated from several responders primed with allogeneic EL4 tumor cells. This indicates that the previously reported results were not unique to the original EL4-primed responder. Second, we establish that peptide-dependent Kb-specific alloreactive CTL clones can be identified after priming with normal allogeneic spleen cells. Thus, induction of peptide-dependent CTL clones is not a unique property of EL4 tumor cells. These results suggest that peptide plays an important role in formation of the allogeneic determinants recognized during graft rejection. Third, these studies reveal that a number of alloreactive CTL clones exhibit cell-type-specific recognition of allogeneic murine target cells. This is consistent with the possibility that some CTL clones recognize peptide antigens that are limited in their tissue distribution. The mechanisms and implications of these findings are discussed.