This study describes an analysis of the interaction of individual amino acid residues of the vesicular stomatitis virus (VSV) nucleocapsid antigenic octapeptide (N52-59; Arg-Gly-Tyr-Val-Tyr-Gln-Gly-Leu) with the H-2Kb molecule and T-cell receptors (TCRs). Tyr-3, Tyr-5, and Leu-8 were the positions in the peptide found to be H-2Kb contact residues by analyzing single alanine-substituted peptides in a competition assay with a Kb-restricted antigenic nonapeptide of Sendai virus. Arg-1, Gly-2, Val-4, Gln-6, and Gly-7 of the peptide were identified as putative TCR contact residues by testing the peptide analogs for their capacity to sensitize targets for VSV-specific cytolytic T-lymphocyte clones. The octamer N52-59 was the optimal length of the peptide required for binding to Kb. This peptide length requirement and the finding of an irregular interspersing of major histocompatibility complex and TCR contact residues are most consistent with the conclusion that the peptide is in an extended conformation in the antigen binding groove. Furthermore, data on binding of truncated peptides show that, although the Arg-1 side chain has been assigned as a TCR contact residue, the main-chain atoms of the N-terminal amino group are most likely involved in interacting with the major histocompatibility complex molecule. A panel of H-2Kb point mutants was constructed to explore the effect of altered amino acid residues on the binding of N52-59. Mutants with amino acid substitutions along the floor of the groove all bound the VSV peptide but modulated its interaction with Kb, apparently causing subtle changes in the spatial arrangement of some specific TCR contact residues in the peptide.