An immunodominant epitope of bovine RNase restricted by I-E(k) molecules was identified using a T cell hybridoma recognizing RNase. This epitope was localized to the peptide RNase(90-105). Single conservative amino acid substitutions were made at each of the positions 94 through 105. It was found that only at one position, Asn-103, were conservative substitutions not allowed. This residue was shown to be the critical residue in determining T cell specificity. The ability of RNase(90-105) and the well-defined T cell epitope, HEL(46-61) to stimulate mouse strains expressing different independent H-2 haplotypes was examined using a T cell proliferation assay. The response to HEL(46-61) was completely restricted to mice expressing an I- A(k) molecule. In striking contrast, 6 of 10 different mouse strains, H- 2(b,f,k,q,s,u), mounted vigorous T cell responses to RNase(90-105). The response was restricted to both I-A and I-E molecules, including I-Ab, I- A(f), I-E(k), I-A(q), and I-A(s). H-2(d) mice were nonresponders to RNase(90- 105), which was shown to be due to the failure of RNase(90-105) to bind to I- A(d) molecules. A variant RNase(90-105) peptide was generated, containing an I-A(d) binding motif, that could bind to I-A(d) molecules. Despite its ability to bind, this variant peptide was not able to stimulate a response in H-2(d) mice. This result demonstrates that the ability of a peptide to bind to an Ia molecule is necessary but not always sufficient for a response to occur. Thus, in contrast to the highly restricted HEL(46-61) determinant, the RNase(90-105) determinant is permissive in its binding to Ia molecules. These results show that in the universe of T cell inducing epitopes contains both highly restricted and broadly restricted epitopes are found.