Ovalbumin-specific, Kb-restricted T cells recognize the minimal fully active synthetic peptide ovalbumin (OVA)257-264. This sequence coincides with the eight residue, allele-specific peptide binding motif previously predicted from direct sequencing of naturally occurring Kb-associated peptides (Falk, K., Rotzscke, O., Stevanovic, S., Jung, G., and Rammensee, H.-G., Nature 351:290, 1990). T cell recognition of a panel of analogs with single residue substitutions between the two putative Kb anchor residues at OVA261 and OVA264 suggested that at least one residue, Glu at position 262, is involved in TCR interaction. OVA-specific cytotoxic T lymphocytes (CTL) derived from TCR beta-chain transgenic mice, where the beta-chain originates from an OVA-specific, Kb-restricted CTL B3, showed that differences in TCR alpha-chain pairing determined the specificity for OVA residue 262. These data support the notion that residue 262 of the OVA T cell determinant, corresponding to position 6 within the Kb-binding motif, represents a contact site for TCR. This residue interacts directly with the TCR alpha-chain or with a site on the TCR beta-chain whose conformation is affected by TCR alpha-chain pairing.