The simian immunodeficiency virus (SIV) macaque model system has been used extensively to study AIDS pathogenesis and to test candidate vaccines for their ability to protect against homologous or heterologous challenge with pathogenic SIV or SHIV. Recent studies suggest that stimulation of HIV-1-specific CTL responses is important for effective vaccination against HIV-1. While quantitative measurements of SIV-specific cytotoxic T lymphocyte (CTL) responses have been facilitated by the use of tetrameric peptide complexes, this technique is currently limited to the study of Mamu-A*01-positive rhesus macaques. Furthermore, very few SIV-specific CTL epitopes have been identified, and there is limited identification of other MHC alleles in macaques. In this study, cytokine flow cytometry (CFC) was used to quantify SIV-specific CD8+ antigen-reactive T cells in macaques infected with SIV. We found a strong correlation (r = 0.96, P