We describe the identification of a heteroclitic determinant that enhances recognition by virus-specific CD8 T cells, and use the crystal structure of the new determinant (S598 Q600Y ) to provide a basis why it elicits an enhanced CTL response. Comparison of S598 to the consensus binding motif suggested a suboptimal interaction with the H-2K b molecule at the secondary anchor position (Gln-3) and, consequently, an approach to enhance the immunogenicity of the determinant. Replacement of the Gln-3 with Tyr-3 (Q600Y) did, indeed, result in an determinant with increased thermostability without diminishing the CD8 T cell response. Most strikingly, the Q600Y change resulted in subtle changes in the conformation of the a-2 helix locally in the vicinity of Glu-152. These subtle changes are likely critical for the enhanced TcR recognition that we detected. S598 Q600Y elicited a response with higher functional avidity to both the cognate and native determinants than S598, and this was not reflected in differential Vb usage. The T cell response to S598 in rJ-infected mice is very diverse [35] . As assessed by Vb usage, the response to S598 and S598 Q600Y in rJ.S598 Q600Y was similarly diverse with only modest differences noted when cells from mice primed by S598 and S598 Q600Y were compared. While we cannot exclude the possibility that cross-reacting S598-specific cells primed by S598 Q600Y are biased for Vb chains not analyzed in this study, it is more likely that the fine specificity of the complementarity-determining region 3 (CDR3) determines their greater affinity for H-2K b /S598. Although an increase in stability of the MHC class I/peptide complex is not generally expected to enhance TcR affinity for the complex, similar results have been observed in mice immunized with analogues to a common tumor antigen [36] .