The nature of alloreactivity to MHC molecules has been enigmatic, primarily because of the observation that allogeneic responses are considerably stronger than syngeneic responses. To better determine the specificity potential of allogeneic responses, we have generated alloreactive CTL specific for exogenous, viral-derived peptide ligands. This approach allowed us to critically evaluate both the peptide- and MHC-specificity of these alloreactive T cells. Exploiting the accessibility of the H-2Ld class I molecule for exogenous peptide ligands, alloreactive CTL were generated that are specific for either murine cytomegalovirus (MCMV) or lymphocytic choriomeningitis virus (LCMV) peptides bound by Ld alloantigens. Peptide specificity was initially observed in bulk cultures of alloreactive CTL only when tested on peptide-sensitized T2.Ld target cells that have defective presentation of endogenous peptides. Subsequent cloning of bulk alloreactive CTL lines generated to MCMV yielded CTL clones that had exquisitely specific MCMV peptide recognition requirement. All of the MCMV/Ld alloreactive CTL clones were also exquisitely MHC-specific in that none of the CTL clones lysed targets expressing MCMV/Lq complexes, even though Lq differs from Ld by only six amino acid residues and Lq also binds the MCMV peptide. This observation clearly demonstrates that alloreactive CTL are capable of the same degree of specificity for target cell recognition as are syngeneic CTL in MHC-restricted responses.