Previous studies have demonstrated that class I major histocompatibility complex (MHC) molecules are occupied by peptides of endogenously synthesized self proteins. Since graft rejection appears to be mediated by the normal occurrence of high frequencies of cytolytic T lymphocytes (CTLs) specific for allogeneic MHC molecules, it is important to know if such CTLs recognize specific MHC-peptide complexes (as opposed to the MHC molecule per se) and, if so, whether allorecognition is the result of the recognition of a limited spectrum of antigenic determinants or, alternatively, the recognition of a diverse array of MHC-self peptide complexes. This issue has been examined using a mutant cell line, T2Kb, that expresses class I molecules devoid of endogenously derived peptides. This cell line was not recognized by Kb-specific alloreactive CTLs. However, upon exposure to peptides derived by cyanogen bromide cleavage of cytoplasmic proteins these cells became sensitized for recognition and lysis by a majority of the CTL clones examined. Reverse-phase HPLC fractionation of the heterogeneous cell-derived peptides revealed that individual CTL clones were specific for different peptide antigen(s). Thus, the high frequency of alloreactive T cells that is responsible for graft rejection appears to represent the sum of numerous T-cell clones specific for a diverse array of endogenous peptide antigens presented in the context of allogeneic class I molecules.