Limit-dilution cultures were used to select vaccinia-immune T-cell populations from bml and bin3 mutant mice that were not lyric for virus-infected targets expressing the K b and Db MHC glycoprotein. Approximately 30% of virus-immune CTL were restricted in each case to K bml and K bin3, rather than to D b. Evidence of extensive cross-reactivity was found for these virus-immune CTL. Bin3 and bml 1 mice sharing one amino acid mutation from wild-type but differing by a second mutation seen only in bin3 are the most cross-reactive pair in their presentation of vaceinia. The bml and bmlO pair with dissimilar mutations from wild-type affecting the same CNBr fragment are also largely cross-reactive. However, 30% cross-reactivity is also found for bml and bin3, which differ in separate CNBr fragments. That mutants expressing amino acid substitutions in the same region of the peptide tend to show more evidence of cross-reactivity does not necessarily mean the T cells see linear arrays of amino acids on the MHC glycoprotein. For instance, K bin1 and K bmm differ for three amino acids, but bin1 T cells are highly lytic for bmIO virus-infected targets. However, there is no cross-reactivity for K bm and K b, which differ at only two amino acids. The key to further understanding may rest with defining the nature of the conformational differences among the K bm~, K bm~Β°, and K b glycoproteins.