Infection of Macaca fascicularis (cynomolgus monkey) with chimeric simian/human immunodeficiency virus (SHIV) provides a valuable experimental animal model of AIDS and is widely used for the development of human immunodeficiency virus vaccine strategies. In these settings, analysis of CD8(+) T-cell responses during infection represents one of the key parameters for monitoring the evaluation of containment of virus replication. The generation of Gag-specific CD8(+) T cells was reported previously from a cynomolgus monkey infected with SHIV89.6P by taking advantage of a B-lymphoblastoid cell line transduced with a retroviral vector expressing simian immunodeficiency virus (SIV) Gag. Here, it was shown that these cytotoxic T lymphocytes (CTLs) demonstrated specificity for a single 9 aa peptide (NCVGDHQAA) spanning aa 192-200 of the SIVmac239 p55(gag) protein. Furthermore, a positive response was found against the same epitope in one of six other SHIV-infected monkeys. This newly identified SIV Gag CTL epitope in SHIV-infected cynomolgus monkeys will be a useful tool for monitoring and evaluating Gag-specific immune responses during vaccination and infection in the cynomolgus monkey model of AIDS.