Single amine acid disparities in MHC class I molecules can elicit transplantation responses. Since β2 microglobulin (β2m) is noncovalently associated with class I antigens on the cell membrane we investigated whether the single amine acid polymorphism at position 85 (Asp → Ale) in the mouse β2m molecule can cause skin graft rejection. A B2mb transgene was introduced into CBA(β2ma) mice which subsequently expressed both forms of β2m. Skin from these CBA β2mb transgenic mice was not rejected by the parental CBA strain. Previous studies showed that cytotoxic T lymphocyte (CTL) responses directed against β2mb use H2K as a restriction element. We therefore produced mice expressing H2Kb and H2Ab as well as β2mb by crossing CBA.β2mb mice with either CBA. Kb (CBK) transgenic mice or C3H.SW mice and used these as skin graft donors for β2mb negative littermates. In both cases rejection of transgenic skin only occurred when mice had received both a β2mb graft and an H2-disparate allograft lying adjacent in the same site. Introduction of the male specific antigen, H-Y, as a helper determinant did not result in rejection of β2mb skin. Neither did two CTL determinants (P91A and β2mb) on the same graft complement one another to elicit a transplantation response. Prior immunisation with tissues expressing the β2m disparity alone did not generate in vive or in vitro β2mb-specific CTL responses, suggesting that this single amino acid difference is not sufficient to elicit a CTL or helper T cell response.