In this study, we have started to dissect the molecular basis of CD8 dependence of a high and low avidity CTL clone specific for the same peptide epitope. Using anti-CD8alpha and anti-CD8beta antibodies, we found that cytotoxicity and IFN-gamma production by high but not by low avidity CTL was strongly CD8 dependent. We isolated the TCR genes of both types of CTL clones and used retroviral gene transfer to analyse the function of these TCR in primary T cells of wild-type and CD8beta-deficient mice. Both TCR triggered antigen-specific killing in wild-type T cells, and blocking experiments showed that CD8 dependence/independence co-transferred with the TCR into primary T cells, indicating that it was dictated by the TCR itself. Gene transfer experiments into CD8beta-deficient T cells revealed that only the TCR derived from the CD8-independent CTL clone elicited antigen-specific cytotoxicity, while the CD8-dependent TCR was non-functional in the absence of the CD8beta-chain. These data indicate a striking difference between CD8alpha/beta heterodimers and CD8alpha/alpha homodimers as only the former were able to provide co-receptor function for the CD8-dependent TCR.