To explore the structural basis for AIDS virus recognition by CD8+ lymphocytes, we sought to determine whether there is a diverse or restricted usage of T-cell receptors (TCR) by simian immunodeficiency virus of macaques (SIVmac) Gag-specific cytotoxic T lymphocytes (CTL) in the rhesus monkey. Six Gag-specific CTL clones were independently generated from an SIVmac-infected rhesus monkey. All six CTL clones recognized a single SIVmac Gag peptide in association with a single major histocompatibility complex class I gene product, Mamu-A*01. TCR alpha-chain sequences from these six CTL clones employed four different V alpha families and five different J alpha gene segments. In contrast, five of the six CTL clones expressed V beta genes that were members of the same family, a human V beta 23 homolog. Furthermore, only one J beta gene was expressed by four of the six CTL clones. These results indicate that TCR of SIVmac Gag-specific CTL from a rhesus monkey can exhibit a restricted usage of V beta gene families and J beta genes.