We report that a peptide from the B chain of insulin, B(10-30), binds with high affinity to multiple class II proteins, including IAb,d,k, IEd,k, and DR1. The ability of B(10-30) to inhibit the binding of other peptide antigens to class II does not correlate with its affinity for class II. B(10-30) only weakly inhibits the binding of antigenic peptides. Conversely, peptides with high affinity for the peptide-binding groove of various class II proteins do not inhibit B(10-30) binding. The rate of association of B(10-30) with class II is unusually rapid, approaching saturation in 1-2 h compared with 1-2 d for classical peptide antigens in the same conditions. The dissociation rate is also relatively rapid. The B(10-30) peptide inhibits the binding of the super-antigen staphylococcal enterotoxin B (SEB) to IAk. It also inhibits SEB-mediated T cell activation. These observations support the conclusion that B(10-30) binds to a site outside the peptide-binding groove. Our findings indicate that short-lived peptide-class II complexes can be formed through interactions involving the SEB-binding site and raise the possibility that alternative complexes may serve as T cell receptor ligands.