We have analyzed the interaction of the hen egg-white lysozyme (HEL) peptide 107-116 with the MHC class II molecule I-Ek, using truncated and single residue substitution analogues to measure activation of I-Ek-restricted, 107-116-specific T cell hybridomas and competition for Ag presentation by I-Ek molecules. These results have been compared with previous findings on the interaction of the same peptide with the I-Ed molecule. Stimulation of T cell hybridomas by truncated peptides defines the sequence 108-116 as the minimum epitope necessary for activation of both I-Ek- and I-Ed-restricted T cell hybridomas. Substitution analysis pinpoints three residues (V109, A110, and K116) in the sequence 108-116 as being critical for binding to I-Ek molecules and demonstrates the involvement of most other residues in recognition by T cells. Results previously obtained for binding of HEL 107-116 to I-Ed molecules indicated that peptide residues R112, R114, and K116 were critical for interaction with I-Ed. Comparison of these results indicates a difference in the likely MHC contact residues between the HEL sequence 108-116 and I-Ed or I-Ek molecules, suggesting that the same HEL peptide assumes a different conformation in the binding site of these two MHC molecules. This in turn affects residues interacting with the specific T cell receptor. According to the hypothetical tridimensional structure predicted for class II molecules, the difference in MHC contact residues observed within the sequence 108-116 can be related to polymorphic amino acids in the binding site of I-Ek and I-Ed molecules. A search through published binding data for a common pattern in this and other I-Ek-binding peptides has permitted us to derive a possible motif for predicting peptide binding to I-Ek molecules. This putative motif was tested by determining binding to I-Ek of an unbiased panel of about 150 synthetic peptides. Binding data indeed demonstrate the presence of this motif in the majority of good binders to I-Ek molecules.