We have investigated the effect of a range of drugs (some commonly coadministered with zidovudine [ZDV] to human immunodeficiency virus-positive patients) on intracellular phosphorylation of ZDV by stimulated peripheral blood mononuclear cells, Molt 4 cells, and U937 cells in vitro. Of the drugs tested (azoles, antiviral agents, antibiotics, and anticancer agents), only doxorubicin and ribavirin caused inhibition of anabolite formation as measured by high-performance liquid chromatography. This in vitro approach may provide important leads to potential interactions at the phosphorylation level in patients with human immunodeficiency virus disease. It is reassuring that so many commonly administered drugs do not alter ZDV phosphorylation.Zidovudine (ZDV), in common with other nucleoside analogs, requires intracellular phosphorylation via ZDV monophosphate (ZDV-MP) and ZDV diphosphate (ZDV-DP) to the active form ZDV triphosphate (ZDV-TP) which competitively inhibits viral reverse transcriptase and results in proviral DNA chain termination (1,13,19,21,23,26).Pharmacokinetic drug-drug interactions involving ZDV may occur at various stages, including absorption, hepatic metabolism (to glucuronide conjugate), and renal excretion of both ZDV and ZDV-glucuronide. Numerous studies have been conducted in these areas and have been recently reviewed (5, 18). However, since intracellular phosphorylation is a key event in the antiretroviral action of ZDV, it is important to design appropriate studies to investigate factors that may modify phosphorylation and hence drug efficacy. In a previous study (28) we demonstrated that didanosine and zalcitabine had no effect on ZDV phosphorylation in vitro in peripheral blood mononuclear cells (PBMCs). In the present work we describe the effect of a number of drugs (antiviral agents, antibiotics, antifungal agents, and others) regularly coadministered with ZDV to human immunodeficiency virus-positive patients on in vitro phosphorylation in activated PBMCs, Molt 4 cells, and U937 cells. A decrease in phosphorylation may have implications for in vivo efficacy of the nucleoside.RPMI medium was purchased from Gibco, Life Technologies Ltd. Penicillin-streptomycin and L-glutamine were obtained from Northumbria Biologicals. Fetal calf serum was acquired from Sera Lab, Sussex, United Kingdom. Mono-poly resolving medium and ribavirin were obtained from ICN Biomedicals Inc., Irvine, Calif. Doxorubicin was obtained from Farmitalia Carlo Erba Ltd., St. Albans, Herts, United Kingdom. Acyclovir, ganciclovir, foscarnet, fluconazole, and ketoconazole and itraconazole were gifts from Wellcome Research Laboratories, Syntex Pharmaceuticals Ltd., Astra Pharmaceuticals, Pfizer Central Research, and Janssen Research Foundation, respectively. All other drugs and chemicals were purchased from Sigma Chemical Co. Ltd.PBMCs were isolated from venous blood obtained from healthy volunteers as previously described (28) and were seeded in 5-cm-diameter petri dishes (3 ϫ 10 6 cells per plate). The mitogen phytohemaggl...