In contrast to T cell recognition of conventional peptide/MHC, T cell recognition of superantigen is not MHC-restricted. However, an influence of MHC polymorphism on specificity is consistent with accumulating data suggesting a TCR/MHC interaction during T cell recognition of superantigen. We have previously shown that T cells from V beta 8.1 beta-chain transgenic mice show an unexpected bias against recognition of Mls-1 presented by H-2d spleen cells. In the current studies we have examined whether thymic positive selection in H-2d mice, which selects T cells that see conventional antigen preferentially in the context of H-2d, is able to overcome the strong bias against recognition of Mls-1/H-2d. The data show that transgenic T cells from both H-2d and H-2k mice have comparable reactivity. The failure of thymic positive selection to overcome the bias against Mls-1/H-2d suggests that the orientation of the putative TCR/MHC interaction during recognition of Mls-1 is not the same as during recognition of conventional peptide/MHC.