To examine the role of peptide in alloreactive class II MHC-restricted responses, we transfected I-A molecules into the Ag-processing defective mutant cell line, T2. Consistent with their defective Ag-processing phenotype, the T2 transfectants predominantly express SDS-unstable I-A molecules on their surface. These cells and phenotypically normal APCs were used to study primary and secondary alloreactive T cell responses in limiting dilution assays. The results demonstrate that the majority of CD4 T cells that participate in primary alloresponses and essentially all the CD4 T cells that participate in secondary alloresponses recognize I-A conformers that depend on the presence of peptide and do not recognize the SDS-unstable I-A expressed by T2 transfectants. To further investigate the requirement for peptide in these responses, we incubated the T2 transfectants with E alpha 52-68 peptide and generated SDS-stable I-A-peptide complexes on the cell surface. The I-Ab-E alpha peptide complexes expressed on T2 cells are stimulatory in secondary alloresponses if the T cells were exposed to the same I-A peptide complex during the priming step. These studies demonstrate that peptide-containing class II MHC is the relevant ligand for alloreactive T cells, and identify an alloreactive response to a peptide (E alpha 52-68) derived from a highly expressed cell surface "self" Ag, the I-E molecule.