Synthetic peptides spanning the entire sequence of both human and mouse beta 2-microglobulin (beta 2M) have been tested for their capacity to bind to three different mouse (I-Ad, I-Ed, and I-Ak) or human (DR1, DR2, and DR5) class II molecules. The results demonstrate that class II molecules do not discriminate between self and non-self peptides. When the immunogenicity of the human beta 2M peptides was measured by their ability to prime H-2d mice for in vitro T cell proliferation, it was found that peptides incapable of binding class II molecules in vitro were also non-immunogenic in vivo. Interestingly, however, several binders, including the human beta 2M peptide 1-16, the best binder in this series to Iad molecules, were found to be non-immunogenic. Since the corresponding mouse beta 2M peptide 1-16 was also capable of binding to Iad molecules, this suggested that lack of responsiveness to the non-self peptide could arise either from central or peripheral tolerance induced by the self homolog. Alternatively, lack of responsiveness could arise from other mechanisms, such as negative selection by other non-homolog sequences or lack of suitable T cell receptor genes. To discriminate between these possibilities, H-2d mice with disrupted beta 2M genes were immunized with the human beta 2M peptide 1-16. This peptide also failed to prime for T cell responsiveness in beta 2M-negative mice, suggesting that a hole in the T cell repertoire for this antigen was not mediated by negative selection or peripheral tolerance induced by self beta 2M peptides.