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 Vβ5.2⁺ 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 α‐ and β‐chains. Closer examination showed that the majority of these sequences encoded negatively and positively charged residues at their respective TCR α‐ and β‐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 K^b‐peptide complexes predict that the major determinant from OVA, peptide OVA_257–264 (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 α‐chain makes contact with P7 lysine, we established a single chain TCR transgenic C57BL/6 mouse line where all T cells express a TCR β‐chain derived from the Vβ5.2⁺ clone B3. OVA‐specific T cells derived from in vivo primed transgenic mice preferentially expressed TCR α‐chains that also contained negatively charged junctional residues despite some further variation in Vα and Jα sequences. Stimulation of naive TCR β‐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 OVA_257–264 determinant, sugesting that the TCR α‐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 α‐ and β‐chains specific for OVA_257–264 and identify their possible orientation over the peptide‐class I complex.