Conflicting data have been reported on ability of 3'-azido-3'-deoxythymidine (AZT) to protect mononuclear phagocytes from HIV-1 infection. We compared the antiviral potency of AZT in three types of primary human mononuclear phagocytes: peripheral blood monocytes, monocyte-derived macrophages (in vitro differentiated) and alveolar macrophages (in vivo differentiated). To establish highly-productive virus infection, purified cells (greater than 99%) from healthy donors were challenged with the macrophage-tropic HTLV-IIIBa-L strain at input multiplicities ranging from 0.05 to 20 TCID50 per cell. AZT (0.1 nM-10 microM) was added immediately after infection and either continued for the duration of the experiment or stopped 1-7 days after infection. The kinetics of HIV-1Ba-L replication were assessed by measuring p24 antigen production on days 4-28 post-infection. Continuous treatment with AZT reproducibly inhibited viral replication in a concentration-dependent manner in all three cell types. The IC90 of AZT was 0.04 microM in blood monocytes, 0.009 microM in monocyte-derived macrophages, and 0.0001 microM in alveolar macrophages (mean of 3-4 donors for each cell type). AZT was not cytotoxic at less than 10 microM as assessed by cell viability, cell protein, and interferon-gamma-activated H2O2-release. In experiments in which AZT treatment was stopped after infection, viral replication resumed after a lag of 7-14 days and increased exponentially toward control levels. This occurred despite initial inhibition of virus production to below the limit of p24 detection (approximately 50 pg/ml). These results indicate that AZT is a potent inhibitor of HIV-1 replication in primary mononuclear phagocytes regardless of the stage of cell differentiation, and that AZT is most active in tissue (alveolar) macrophages. AZT does not irreversibly block infection of mononuclear phagocytes, however, as viral replication resumes after removal of AZT.