A transgenic mouse has been made that expresses a mutant MHC class I H-2Kb molecule with glutamic acid at position 65 (E65) in place of glutamine. The side chain at position 65, on the outward face of the alpha-helix of the alpha 1 domain of the class I molecule, interacts with the TCR, and not with the peptide binding groove. The transgenic mouse, on a DBA/2 background, mounts Kb,E65-restricted Ag-specific responses to conventional Kb-restricted Ag such as OVA and vesicular stomatitis virus, and shows strong alloreactivity to wild-type Kb. The transgenic mouse also mounts a primary in vitro alloreactive response directed to a mutant molecule with aspartic acid at position 65 (D65). This response is relatively weak, probably because of the structural similarities between aspartic and glutamic acid side chains; both have carboxylic termini, and the aspartic acid side chain is shorter by a single secondary carbon. The alloreactive CTL lines elicited by this conservative change are cross-reactive among several position-65 variants of H-2Kb. Individual CTL clones are specific for self peptides that can be extracted from cells expressing Kb,E65, and from purified wild-type Kb molecules, and that are recognized in the context of the D65 residue. Thus, the smallest variance from self in a class I molecule, even outside the peptide binding groove, can be antigenic.