Answers to questions about frequency and repertoire of immune cells, relative contributions made by different types of immune cells toward the total Epstein-Barr virus (EBV)-directed response and the variation of such responses in healthy persons have been elusive because of disparities in assays, antigen presenting cells, and antigenic sources used in previous experiments. In this study, we addressed these questions using an assay that allowed direct comparison of responses generated by different types of cells of the immune system. This short-term (20-hour) ex vivo assay measured interferon-gamma production by blood cells in response to autologous EBV-transformed lymphoblastoid cell lines (LCLs). Our experiments defined the variation in responses among persons and clearly distinguished 10 healthy EBV-immune from 10 healthy EBV-naive persons. In EBV-immune persons, 33% of responding cells were CD4(+), 43.3% were CD8(+), and 12.9% were gamma-delta T cells. LCL-reactive CD8(+) T cells were only 1.7-fold more frequent than similarly reactive CD4(+)T cells. Responses by gamma-delta T cells were 6-fold higher in seropositive than in seronegative persons. Our findings emphasize the importance of CD4(+) and gamma-delta T-cell responses and have implications for immunotherapy and for identifying defects in T-cell populations that might predispose to development of EBV-associated lymphomas.