Objectives and designMeasurement of phosphorylated zidovudine (ZDV) inside infected cells is more likely to provide satisfactory dose response relationships than serum concentrations. This study provides information on ZDV phosphorylation in HIV-seronegative volunteers (n = 5) and in patients with HIV infection (n = 12).Methods:Intracellular ZDV phosphate metabolites were measured in peripheral blood mononuclear cells isolated from whole blood by density cushion centrifugation. Cells were washed and extracted overnight with 60% methanol prior to analysis by high performance liquid chromatography. Fractions eluted from the column corresponding to ZDV, ZDV monophosphate (ZDV-MP), ZDV diphosphate (ZDV-DP) and ZDV triphosphate (ZDV-TP) were collected, hydrolysed by acid phosphatase and ZDV levels quantified by radioimmunoassay.Results:The area under the plasma ZDV concentration-time curve (AUC06–6h) was similar in seronegative volunteers and patients [mean ± SD, 4.64 ±2.50 versus 5.56±2.67 μmoles/l h; 95% confidence interval (Cl, −4.39–2.23;P=0.646, Mann-Whitney U test]. However, ZDV phosphorylation was greater in patients, with the AUC0–6hfor total phosphate metabolites being 5.91 ±3.42 pmoles/106cells h compared with seronegative volunteers (0.66±0.48 pmoles/106cells h; 95% Cl, −8.35 to –2.32;P= 0.0003). The concentration of ZDV-TP was similar in both groups, the increase in total phosphates in patients being due primarily to ZDV-MP. ZDV-MP AUC0–6hand total ZDV phosphate AUC0–6hwere closely correlated (r2= 0.94). The relationship between total ZDV phosphate AUC0–6hand the CD4 count demonstrates that patients with a count < 100×106/l have much higher ZDV phosphate levels, predominantly ZDV-MP.Conclusion:ZDV is phosphorylated to a greater extent in patients than in healthy volunteers. The increased ZDV-MP in patients with low CD4 counts may explain the well known occurrence of increased ZDV toxicity in patients with more advanced disease. The ability to measure ZDV phosphorylated metabolites (without the administration of radiolabelled nucleoside) represents a significant advance in our understanding of the clinical pharmacology of the drug.