MHC class I-restricted CTL play an important role in limiting the spread of HIV-1 in the infected individual. Elucidating the molecular interactions of CTL with the virus is, therefore, of central importance for characterizing the immune control of this infection. In exploring this CTL response, we have defined the TCR usage by SIVmac Gag-specific CTL in rhesus monkeys. Thirty-nine CTL clones were generated from PBL of three SIVmac-infected monkeys expressing the MHC class I Mamu-A*01 gene product, all of which were shown to recognize a single SIVmac Gag peptide in association with Mamu-A*01. Sixty-six percent of CTL clones derived from two monkeys early after infection expressed TCR genes of the V beta 13 family; 70% of these V beta 13+ CTL clones expressed a TCR heterodimer composed of V alpha 1 and V beta 13 gene products. In addition, there appeared to be a selection of a single conserved amino acid and restricted CDR3 lengths in junctional regions of TCR beta-chains expressed by the V beta 13+ CTL clones. These findings indicate significant structural constraints on the CTL-TCR interaction with the AIDS virus. Interestingly, 55% of the CTL clones derived from the third animal at a later time following infection employed genes of the V beta 6 family in their TCR. Despite the preferential use of TCR V family genes by the CTL clones, the SIVmac Gag-specific CTL response was clearly polyclonal; TCR expressed by these CTL clones displayed varied sequences in their CDR3 regions. Other V gene families, including V beta 23, V alpha 8, and V alpha 20, were used in TCR expressed by SIVmac Gag-specific CTL clones. These studies, therefore, indicate that the TCR repertoire of SIVmac Gag-specific CTL that share a peptide and MHC class I recognition specificity can be diverse. Such a broad CTL-TCR repertoire may be advantageous for the host in containing an AIDS virus infection.