CD8 T cells drive the protective immune response to lymphocytic choriomeningitis virus (LCMV) infection and are thus a determining force in the selection of viral variants. To examine how escape mutations affect the presentation and recognition of overlapping T-cell epitopes, we isolated an LCMV variant that is not recognized by T-cell receptor (TCR)-transgenic H-2D^b-restricted LCMV GP33-41-specific cytotoxic T lymphocytes (CTL). The variant virus carried a single-amino-acid substitution (valine to alanine) at position 35 of the viral glycoprotein. This region of the LCMV glycoprotein encodes both the D^b-restricted GP33-43 epitope and a second epitope (GP34-42) presented by the K^b molecule. We determined that the V-to-A CTL escape mutant failed to induce a D^b GP33-43-specific CTL response and that D^b-restricted GP33-43-specific CTL induced by the wild-type LCMV strain were unable to kill target cells infected with the variant LCMV strain. In contrast, the K^b-restricted response was much less affected. We found that the V-to-A substitution severely impaired peptide binding to D^b but not to K^b molecules. Strikingly, the V-to-A mutation did not change any of the anchor residues, and the dramatic effect on binding was therefore unexpected. The strong decrease in D^b binding explains why the variant virus escapes the D^bGP33-43-specific response but still elicits the K^b-restricted response. These findings also illustrate that mutations within regions encoding overlapping T-cell epitopes can differentially affect the presentation and recognition of individual epitopes.