The P91A antigen was identified following mutation of P1 mastocytoma cells. The peptide epitope is encoded by a mutant form of the S3 subunit of the PA700 proteasome regulatory complex. P91A stimulates a strong CD8+ T cell response when expressed on tumor cells or normal tissue and P91A-specific T cells express a restricted range of T cell receptors. Although it is a strong Ld-binding peptide, P91A does not conform to the established motif for this major histocompatibility complex (MHC) molecule and this has hampered elucidation of the precise epitope. Ld predominantly associates with nonamer peptides; however, using a variety of complementary approaches, the P91A epitope is identified as the octamer QNHRALDL. In the absence of the Ld motif residue proline at position 2, residues 5-7 are primarily involved in MHC interaction. P91A is thus atypical in its interaction with Ld. Residues 1, 3, and 4 are found to influence T cell recognition of P91A. Definition of the P91A peptide will allow studies on P91A processing and interactions of the P91A peptide/MHC complex with T cell receptors of differing avidity to establish the basis for restricted T cell receptor usage. The basis for the failure of the P91A tum+ peptide (QNRRALDL) to bind to Ld is addressed by molecular modeling.