CMV-specific CD4+ and CD8+ T cell IFNgamma expression and proliferation were measured in healthy volunteers by flow cytometry after CMV lysate or CMV pp65 or IE peptide pool stimulation. Cutoff values were set to maximize specificity (i.e., no false positive CMV-seronegatives). Sensitivity (defined as a positive response in CMV-seropositives to at least one of the 3 antigen preparations used) was 100% for CMV-specific CD4+ and CD8+ T cell IFN expression and CD4+ T cell proliferation and 95.4% for CMV-specific CD8+ T cell proliferation. All 22 CMV-seropositive individuals had positive responses by at least three of these four measurements. These findings support the concept that a multiplicity of antigen-specific functional immune responses and persistence of robust virus-specific CD4+T cells are important components of protective immunity in this chronic viral infection.