An individual's T lymphocytes are highly reactive to allogeneic MHC molecules. As a step in deciphering the mechanism of allorecognition by T lymphocytes, we have attempted to identify the TCR's target on MHC class II molecules, in particular the polymorphic residues that determine the specificity of recognition. We have generated a panel of Ak-reactive, Ab-nonreactive T cell hybridomas, and sets of L cell transfectants displaying A alpha A beta molecules with wild-type, chimeric or single site-mutated A alpha chains, with reciprocal interchanges between Ak and Ab. We then measured the stimulation of the T hybridomas in response to the transfectants. The results indicate that the hybridomas recognize diverse and complex determinants, with contributions from both A alpha and A beta chains, and from several regions or amino acids of the A alpha chain. The data are most consistent with a model in which alloreactivity results from the presentation of peptides to the T cell by an allogeneic MHC molecule, peptides that cannot be presented by the responder's own MHC complexes. The specificity of allorecognition seems to be imparted mainly by peptide/MHC molecule rather than TCR/MHC molecule contacts.