We have recently described a CD8+ T-cell clone recognizing defined epitopes of both mycobacterial and murine hsp60 that are not sequence homologues. Adoptive transfer of this T-cell clone into T-cell deficient mice induced an autoimmune intestinal pathology. TCR analysis revealed the productive in frame rearrangement of two TCRa genes in this clone. Expression of two different TCR α chains by one T cell (dual TCR) is discussed as a potential mechanism underlying T-cell mediated autoimmunity. Here we addressed the question of whether hsp60 crossrecognition of self and non-self origin is directly linked to the surface expression of two TCRs by the same cell. Consequently, the potentially dual TCR of the hsp60 reactive T-cell clone was dissected into two single TCRs by double retroviral transduction of TCR deficient cell lines. Our data show that only one of the two TCR α/β combinations formed a functional cell surface TCR and that post-translational allelic exclusion of the second α chain was achieved by the inability to pair with the TCR β chain. Thus a single TCR is not only sufficient for crossrecognition with peptides that share minimal sequence homology, moreover this promiscuous TCR reactivity accounts also for immunopathology as recently shown.