The effects of thymidine and uridine on the phosphorylation of 3'-azido-3'-deoxythymidine (AZT) were studied in various human mononuclear cell preparations. Thymidine suppressed [3H]AZT phosphorylation in the same concentration range (20 to 100 ,uM) in which it antagonizes the anti-human immunodeficiency virus activity of AZT. Uridine, in turn, had no influence on AZT phosphorylation, just as it has no effect on the anti-human immunodeficiency virus activity of AZT. These findings are consistent with a close relationship between the inhibition of AZT phosphorylation and the influence of physiological nucleosides on the antiviral activity of AZT.The antiviral activities of 3'-azido-3'-deoxythymidine (AZT; zidovudine) and other 2',3'-dideoxynucleoside drugs in human immunodeficiency virus (HIV)-infected cells is thought to depend on three main factors: (i) the efficiency of phosphorylation to the triphosphate form, (ii) the efficiency with which the triphosphates inhibit viral reverse transcriptase, and (iii) the influence of physiological nucleosides on the former two processes (8). Concerning the last effect, it has been shown in ATH8 cells (9) and peripheral blood mononuclear cells (PBMC) (11) that thymidine (dThd) counteracts the antiviral activity of AZT. Uridine (Urd), on the other hand, was found not to affect the antiviral activity of AZT, although it antagonized the cytotoxic effect of AZT on bone marrow progenitor cells in a granulocyte-macrophage CFU assay (11). On this basis, Urd was suggested to have potential use in the treatment of human immunodeficiency virus (HIV) infections as a rescue from the hematologic side effects of AZT (11).To help understand the molecular basis of the abovedescribed phenomena, we asked in the present study whether the different effects of dThd and Urd on the anti-HIV action of AZT in PBMC and on the cytotoxicity of AZT in bone marrow mononuclear cells (BMMC) could be correlated with the effects of these nucleosides on the phosphorylation of AZT in these cells.Since monocytes constitute 20 to 30% of PBMC and since nucleoside metabolism is thought to differ between lymphoid cells and monocyte-macrophage lineage cells, purified monocytes and monocyte-derived macrophages (M/M) were subjected to separate analyses as well.PBMC were prepared from blood freshly drawn from healthy volunteers, and BMMC were prepared from a bone marrow aspirate from a healthy donor by Ficoll-Paque gradient centrifugation (5). Washed mononuclear cells were suspended in Dulbecco's modified Eagle's medium supplemented with L-glutamine, 10% heat-inactivated fetal calf serum, 4.5 g of D-glucose per liter, and antibiotics (D-MEM). Monocytes were separated from PBMC by counterflow centrifugal elutriation (16,17) and suspended in D-MEM (-3 x 107/ml). To prepare M/M, we plated monocytes in six-well * Corresponding author.Costar plates (5 x 106 cells per well) and cultured them for 1 day before starting the experiment.[3H]AZT (20 Ci/mmol) was from Moravek Biochemicals (Brea, Calif.). To measure AZT phosphoryl...