We have analyzed the T cell receptor (TCR) repertoire found in the major histocompatibility complex class I-restricted cytotoxic T lymphocyte (CTL) response to the protein ovalbumin (OVA). Despite skewing towards the expression of V beta 5.2+TCR by OVA-specific CTL from C57BL/6 mice, we found a relatively high degree of diversity in V(D)J usage in both TCR alpha- and beta-chains. Closer examination showed that the majority of these sequences encoded negatively and positively charged residues at their respective TCR alpha- and beta-chain VJ or VDJ junctions. These junctions form the third complementarity-determining regions (CDR3) of the TCR polypeptides involved in the direct interaction with the class I-bound peptide. Crystallographic analyses of Kb-peptide complexes predict that the major determinant from OVA, peptide OVA257-264 (SIINFEKL), contains two exposed charged side chains which can contact the TCR. These are the negatively charged glutamic acid at determinant position 6 (P6) and the positively charged lysine at P7. To examine whether the TCR alpha-chain makes contact with P7 lysine, we established a single chain TCR transgenic C57BL/6 mouse line where all T cells express a TCR beta-chain derived from the V beta 5.2+ clone B3. OVA-specific T cells derived from in vivo primed transgenic mice preferentially expressed TCR alpha-chains that also contained negatively charged junctional residues despite some further variation in V alpha and J alpha sequences. Stimulation of naive TCR beta-chain transgenic T cells with a P7 substitution peptide analogue induced a T cell response that was no longer cross-reactive with the wild-type OVA257-264 determinant, suggesting that the TCR alpha-chain from the T cell clone B3 can determine the specificity for this residue. Consequently, these results reveal the existence of conserved residues in the CDR3 of TCR alpha- and beta-chains specific for OVA257-264 and identify their possible orientation over the peptide-class I complex.