We have studied the interaction of six photoreactive conjugates of the immunogenic hen egg-white lysozyme peptides HEL(46-61) or HEL(49-61) with the murine histocompatibility class II molecules I-Ak, I-Ad, I-Ek, and I-Ed. All compounds tested selectively labeled the alpha chain of the class II molecules. This was true when testing class II molecules on cell membranes or solubilized in detergents. The COOH-terminal conjugate of HEL(49-61) with (4-azidobenzoyl)cystine preferentially labeled I-Ak. However, addition of hydroxyl or iodine substituents to the photoreactive moiety increased the labeling efficiency and resulted in labeling of the other class II molecules. The data suggest that the photoreactive groups enhanced the binding affinities of these peptides to class II molecules, reflected by the increased labeling efficiencies. Conversely, introduction of an iodine substitution into the tyrosine residue of HEL(46-61) or HEL(49-61) strongly decreased the photoaffinity labeling, possibly due to steric interference with ligand binding to class II molecules. Judicious use of photoaffinity probes that conserve binding specificity of the peptide should be useful for mapping the antigen-binding site of a class II molecule.