The thymus leukemia antigens (TL) belong to the MHC class Ib family and can be recognized by CD8-dependent or -independent cytotoxic T lymphocytes (CTL) showing TL, but not H-2, restriction. We previously reported that the CTL epitope is TAP independent and in the present study we further characterize the recognition mechanism of CD8-dependent TL-specific TCRalphabeta CTL. We first prepared empty TL tetramers by way of peptide-independent folding with recombinant proteins produced in an Escherichia coli expression system, and showed that TL-specific CTL recognized TL without putative TL-associated peptide and/or post-translational modifications of TL by mammalian and insect cells. We next prepared transfectants; expressing various chimeric TL molecules with mouse or human MHC class I as well as chimeric TL tetramers with recombinant proteins produced by insect cells, and demonstrated that chimeric TL whose alpha3 domain was replaced by that of H-2K(b), but not of HLA-A2, was sufficient for binding and activation of TL-specific CTL. These results indicate that TL-specific CTL recognize predominantly their alpha1/alpha2 domain as an epitope(s) and that the binding activity to the murine CD8 of the alpha3 domain of H-2K(b) is sufficient to induce their CTL activity, although it is known to be weaker than that of TL, but stronger than that of HLA. The results taken together indicate that CD8-dependent TL-specific TCRalphabeta CTL recognize an epitope(s) of the alpha1/alpha2 domain of TL free from antigenic molecules, and that CD8 plays an important role in stable interactions between TL and their corresponding TCR.