OBJECTIVE It has been previously shown that zidovudine (ZDV) and its phosphorylated metabolites can be chemically reduced into the corresponding stavudine (d4T) forms in solution. The aim of this study was to search for intracellular d4T-triphosphate (TP) in patients receiving ZDV therapy as part of highly active antiretroviral therapy and to examine the ratio of concentrations of d4T-TP : ZDV-TP in these patients. METHODS Seven ml of blood were sampled between 0.5 and 13.7 h after the last ZDV dosing in 31 patients. Peripheral blood mononuclear cells (PBMC) were separated using Vacutainer CPT tubes. Intracellular d4T-TP and ZDV-TP concentrations were determined by a newly developed high performance liquid chromatography/tandem mass spectrometry method. RESULTS Intracellular d4T-TP was found in all ZDV-treated patients. d4T-TP concentrations ranged between 3 and 38.5 fmol/1 x 10 cells and represented between 0.03 and 0.37 of the corresponding ZDV-TP concentrations. These d4T-TP concentrations are in the lower range of those measured in d4T-treated patients. The intracellular transformation of ZDV into d4T-TP was also observed during experiments in cells cultured in the presence of ZDV. d4T-TP was never detected in PBMC from patients treated with neither ZDV nor d4T. CONCLUSIONS Significant levels of d4T-TP can be measured in PBMC from patients receiving ZDV therapy. This observation sheds new light on the cross resistance observed between ZDV and d4T and indicates that, in patients treated with ZDV, d4T-TP could participate in the antiretroviral activity and/or toxicity of the drug.