The replication of human cytomegalovirus (HCMV) strain AD169 was studied in human peripheral blood granulocytes, monocytes-macrophages, B lymphocytes, and T lymphocytes. Progeny virus was produced in some T-cell cultures stimulated in the allogeneic mixed lymphocyte reaction and was regularly obtained when stimulated T cells were grown in the presence of interleukin 2. Replication of HCMV in these cultures was documented by increases in titer, expression of early and late antigen as assessed by indirect immunofluorescence and Western blot, and viral DNA synthesis as determined by dot-blot assays. Approximately 0.05% of cells in virus-producing cultures formed infectious centers, indicating that only a subset of cells takes part in active virus replication. In double-immunofluorescence experiments this subset was found to consist primarily of the T3+ and T8+ phenotype. By infection of preparatively separated T4+ and T8+ T lymphocytes, however, it could be shown that both T-cell subsets were susceptible to HCMV infection as indicated by increases in titer and by DNA kinetics. We conclude from these data that the T lymphocyte might be a target for HCMV in vitro, which is in accordance with in vivo findings in HCMV-infected patients.