Nitrotyrosine is widely recognized as a surrogate marker of up-regulated inducible NO synthase expression at sites of inflammation. However, the potential immunogenicity of autologous proteins containing nitrotyrosine has not previously been investigated. Herein, we used the IE K-restricted T cell epitope of pigeon/moth cytochrome c (PCC/MCC 88–103) to assess the ability of T cells to recognize ligands containing nitrotyrosine. Substitution of the single tyrosine (Y97) in PCC/MCC 88–103 with nitrotyrosine abrogates recognition by the MCC 88–103-specific T cell hybridoma 2B4. CBA (H2 K) mice immunized with MCC 88–103 or nitrated MCC 88–103 peptides produce T cell responses that are mutually exclusive. Transgenic mice that constitutively express PCC under the control of an MHC class I promoter are tolerant toward immunization with MCC 88–103, but exhibited a robust immune response against nitrated MCC 88–103. Analysis of T cell hybridomas specific for nitrated-MCC 88–103 indicated that subtle differences in TCR VDJ gene usage are sufficient to allow nitrotyrosine-specific T cells to escape the processes of central tolerance.