Antigen-specific T cells may be detected and enumerated by short-term ex vivo antigen-specific stimulation followed by cytokine flow cytometry. Most frequently, intracellular IFN-gamma is used to identify T cells specific for cytomegalovirus (CMV), Epstein-Barr virus or HIV. Some researchers use whole blood, others peripheral blood mononuclear cells (PBMC) in this assay; however, the performance of the two systems has never been directly compared. Blood was drawn from previously characterized healthy CMV-positive donors, and CMV-derived peptides or CMV lysate were used as stimulants. In an initial series of experiments, lithium-heparin was identified as the best coagulant to be used. Dose-response curves were established using concentrations between 0.1 and 40 microg/ml of peptides and between 0.1 and 20 microg/ml of virus lysate, respectively. IFN-gamma-positive T cells were expressed as percent of the reference population, and frequencies measured in whole blood and PBMC were compared. Maximum responses were consistently higher in PBMC than in whole blood and were reached at lower concentrations of stimulant. In several instances, responses identified with PBMC were not at all detected with whole blood. In summary, studies using whole blood in this type of assay are likely to underestimate the frequencies of antigen-specific T cells.