Immunohistochemical detection of increased levels of protein-associated nitrotyrosine has become widely used as a surrogate marker of in situ inflammation. However, the potential consequences of protein-associated nitrotyrosine formation in terms of cellular immune recognition has received surprisingly little attention. Using a well-defined IE K-restricted epitope of pigeon cytochrome c, we previously demonstrated that conversion of a single tyrosine residue to nitrotyrosine can have a profound effect on recognition by CD4 T cells. In this study, we used the MHC class I-restricted epitope of lymphocytic choriomeningitis virus glycoprotein (gp33) to demonstrate that conversion of tyrosine to nitrotyrosine can also profoundly affect recognition of MHC class I-restricted epitopes. Conversion of the Y4 residue of the gp33 epitope to nitrotyrosine completely abrogated recognition by gp33-specific T cells from P14 TCR-transgenic mice. In contrast, CD8+ T cells specific for “nitrated gp33”(NY-gp33) can be readily elicited in C57BL/6 mice after immunization with NY-gp33 peptide. Interestingly, TT hybridomas specific for NY-gp33 peptide were found to fall into two distinct subsets, being specific for NY-gp33 presented in the context of either H-2D b or H-2K b. This latter result is surprising in light of previous structural studies showing that Y4 comprises a critical TCR-contact residue when presented by H-2D b but that the same residue points downward into the peptide-binding groove of the MHC when presented by H-2K b. Together, these results indicate that nitrotyrosine formation can impact T cell recognition both directly, through alteration of TCR-contact residues, or indirectly, through alterations in MHC-contact positions.