The murine T cell response to bacteriophage lambda cI repressor protein has been investigated. Isolation and characterization of class II-restricted T cell hybridomas from BALB/c and A/J mice undergoing a primary response has revealed that a single region of the protein, residues 12-26, is the immunodominant site. Fine specificity analysis using truncated peptides (P12-24 and P15-26) reveals a great deal of heterogeneity at the clonal level of I-Ad-restricted T cells. I-Ek-restricted T cells are less heterogeneous in their reactivity toward P12-24 and P15-26, but show diversity in their responses to peptide analogues with substitution at Tyr22. The specificity difference between T cell hybridomas of I-Ad-restriction and I-Ek-restriction and the inhibition effect of different inactive peptides suggest that the same peptide is presented in different configurations by different Ia molecules. Further, no cross-reactivity can be detected between T cells of these two haplotypes, Ia molecules and Ia bound-peptides.