Because of the probable role of HIV-infected monocyte/macrophages in the pathogenesis and progression of AIDS, it is essential that antiretroviral therapy address viral replication in cells of this lineage. Several dideoxynucleosides have been shown to have potent in vitro and, in the case of 3'-azido-2',3'-dideoxythymidine (AZT) and 2',3'-dideoxycytidine (ddC), in vivo activity against HIV. However, because these compounds must be phosphorylated (activated) in target cells, and because monocyte/macrophages may have levels of kinases that differ from those in lymphocytes, we investigated the capacity of these drugs to suppress HIV replication in monocyte/macrophages using HIV-1/HTLV-IIIBa-L (a monocytotropic isolate). In the present study, we observed that HTLV-IIIBa-L replication in fresh human peripheral blood monocyte/macrophages was suppressed by each of three dideoxynucleosides: 3'-azido-2',3'-dideoxythymidine (AZT), 2',3'-dideoxycytidine (ddC), and 2',3'-dideoxyadenosine (ddA). Similar results were observed in 5-d-cultured monocyte/macrophages, although higher concentrations of the drugs were required. We then studied the metabolism of AZT and ddC in such cells. The phosphorylation of ddC to a triphosphate moiety was somewhat decreased in monocyte/macrophages as compared with H9 T cells. On the other hand, the phosphorylation of AZT in monocyte/macrophages was markedly decreased to 25% or less of the level in T cells. However, when we examined the level of the normal endogenous 2'-deoxynucleoside triphosphate pools, which compete with 2',3'-dideoxynucleoside triphosphate for viral reverse transcriptase, we found that the level of 2'-deoxycytidine-triphosphate (dCTP) was six- to eightfold reduced, and that of 2'-deoxythymidine-triphosphate (dTTP) was only a small fraction of that found in T cell lines. These results suggest that the ratio of dideoxynucleoside triphosphate to normal deoxynucleoside triphosphate is a crucial factor in determining the antiviral activity of dideoxynucleosides in HIV target cells, and that the lower levels of dTTP may account for the antiretroviral activity of AZT in the face of inefficient phosphorylation of this compound.