ABSTRACT Intracellular phosphorylation of stavudine (d4T) and zidovudine (ZDV) was investigated in peripheral blood mononuclear cells (PBMCs) isolated from ZDV-naive and ZDV-experienced human immunodeficiency virus (HIV)-positive patients. An in vivo study measured the amount of d4T triphosphate (d4TTP), while an ex vivo study assessed the capacity of cells to phosphorylate added d4T. Endogenous dTTP was also measured. d4TTP and dTTP were determined in vivo using a reverse transcriptase chain termination assay. In ex vivo studies, d4T (1 μM) was incubated in resting and phytohemagglutinin-stimulated (10 μg ml −1 ; 72 h) PBMCs for 24 h. After washing and methanol extraction, radiolabeled anabolites were detected by high-performance liquid chromatography. d4TTP reached its highest level 2 to 4 h after dosing (0.21 ± 0.14 pmol/10 6 cells; n = 27 [mean ± standard deviation]). Comparison of ZDV-naive and ZDV-experienced individuals showed no significant difference in levels of d4TTP (ZDV naive, 0.23 ± 0.17 pmol/10 6 cells [ n = 7] versus ZDV experienced, 0.20 ± 0.14 pmol/10 6 cells [ n = 20]; P = 0.473) or the d4TTP/dTTP ratio (0.14 ± 0.12 [ n = 7] and 0.10 ± 0.08 [ n = 20], respectively; p = 0.391). Ex vivo data demonstrated no significant difference in the formation of d4TTP or total d4T phosphates in naive and experienced patients (0.086 ± 0.055 pmol/10 6 cells in ZDV-naive patients [ n = 17] versus 0.081 ± 0.038 pmol/10 6 cells in ZDV-experienced patients [ n = 22]; P = 0.767). The ability of HIV-infected patients to phosphorylate d4T in vivo and ex vivo was unchanged with increasing exposure to ZDV.