Activation of the anti-human immunodeficiency virus (HIV) compound 3'-azido-3'-deoxythymidine (AZT) is dependent on its 5'-phosphorylation by cellular nucleoside and nucleotide kinases. Azidothymidine 5'-triphosphate (AZTTP) is considered to be the metabolite responsible for both the anti-HIV effect of AZT, via inhibition of reverse transcriptase, and cytoxicity by interference with cellular DNA polymerases. During the characterization of AZT metabolism in cultured human T-lymphoblastoid CEM cells, a spontaneously occurring variant cell line, CEM/Ag-1, was found that showed approximately 10-fold resistance to AZT growth inhibition as compared to wild type (wt) cells (EC50 = 2 mM as compared to 350 microM for wt cells). CEM/Ag-1 cells had a 3-fold reduced capacity to accumulate azidothymidine monophosphate (AZTMP) compared to wt cells whereas similar levels of AZTTP were found in both cell lines. The intracellular half-life of AZTMP was approximately 70 min in both wt and CEM/Ag-1 cells. A 3-fold lower specific activity of cytoplasmic thymidine kinase was observed in CEM/Ag-1 extracts as compared to wt. The reduced thymidine kinase activity was not correlated to a decreased level of thymidine kinase mRNA. Syncytium formation of CEM/Ag-1 cells infected with HIV-2 as well as HIV-1 antigen production was inhibited at the same concentrations of AZT (approx. 0.01 microM) as were HIV-1 and HIV-2 infected wt cells. Thus, minor decreases in cellular thymidine kinase levels may markedly affect the cytoxicity of AZT but have no major effect on the antiviral activity of AZT. Our results strongly suggest that AZTMP is responsible for a major part of the growth inhibitor effects, while AZTTP mainly mediates the antiviral activity of AZT.