The frequency of cytomegalovirus (CMV)-specific CD4⁺ T lymphocytes was determined in CMV-seropositive rhesus macaques with or without simian immunodeficiency virus (SIV) infection by using the sensitive assays of intracellular cytokine staining and gamma interferon ELISPOT. Both techniques yielded 3- to 1,000-fold-higher frequencies of CMV-specific CD4⁺ T lymphocytes than traditional proliferative limiting dilution assays. The median frequency of CMV-specific CD4⁺ T lymphocytes in 23 CMV-seropositive SIV-negative macaques was 0.63% (range, 0.16 to 5.8%). The majority of CMV-specific CD4⁺ T lymphocytes were CD95^pos and CD27^lo but expressed variable levels of CD45RA. A significant reduction (P < 0.05) in the frequency of CMV-specific CD4⁺ T lymphocytes was observed in pathogenic SIV-infected macaques but not in macaques infected with live attenuated strains of SIV. CMV-specific CD4⁺ T lymphocytes were not detected in six of nine pathogenic SIV-infected rhesus macaques. CMV DNA was detected in the plasma of four of six of these macaques but in no animal with detectable CMV-specific CD4⁺ T lymphocytes. In pathogenic SIV-infected macaques, loss of CMV-specific CD4⁺ T lymphocytes was not predicted by the severity of CD4⁺ T lymphocytopenia. Neither was it predicted by the pre-SIV infection frequencies of CD45RA^neg or CCR5^pos CMV-specific CD4⁺ T lymphocytes. However, the magnitude of activation, as evidenced by the intensity of CD40L expression on CMV-specific CD4⁺ T lymphocytes pre-SIV infection, was three- to sevenfold greater in the two macaques that subsequently lost these cells after SIV infection than in the two macaques that retained CMV-specific CD4⁺ T lymphocytes post-SIV infection. Future longitudinal studies with these techniques will facilitate the study of CMV pathogenesis in AIDS.