The precise measurement of epitope-specific cytotoxic T lymphocyte (CTL) responses in simian immunodeficiency virus (SIV)- and simian-human immunodeficiency virus (SHIV)-infected or vaccinated rhesus monkeys has been important in the evaluation of potential HIV vaccine strategies. This quantitation of CTL has been limited to date by the identification of only one dominant SIV/SHIV epitope in these monkeys. We have recently defined a Nef CTL epitope p199RY (YTSGPGIRY) that is recognized by CD8(+) T lymphocytes from all SIV/SHIV-infected Mamu-A*02(+) rhesus monkeys that have been evaluated. We now measure the frequency of p199RY-specific CD8(+) T lymphocytes in the peripheral blood of these monkeys with quantitative precision, using MHC class I/peptide tetramer staining and peptide-stimulated interferon-gamma Elispot assays. These epitope-specific CD8(+) T lymphocytes are present at a very high frequency and represent a significant proportion of the entire SIV- or SHIV-specific CD8(+) T lymphocyte population in SIV/SHIV-infected Mamu-A*02(+) rhesus monkeys. Knowledge of this dominant CTL epitope should prove valuable in the evaluation of HIV vaccine strategies using this animal model.