An assay to quantify the phosphorylation products of zidovudine (AZT) in peripheral blood mononuclear cells (PBMC) was developed. Extracts of PBMC were separated by high-performance liquid chromatography. Eluted AZT mono- (MP), di- (DP), and triphosphate (TP) were collected in separate portions. Treatment with alkaline phosphatase yielded equimolar amounts of AZT, which after solid-phase enrichment were assayed by radioimmunoassay. Detection limit was 0.1 pmol/10(6) PBMC for each nucleotide. Recoveries of 102%-118% were observed. AZT nucleotides were measured in samples from three patients receiving 250 mg of AZT every 12 h. Intracellular concentrations of AZT-MP after 1-2 h ranged from 0.9 to 1.4 pmol/10(6) PBMC and then declined to 0.3-1.1 pmol/10(6) PBMC after 4 h. AZT-DP and AZT-TP reached concentrations of 0.3-0.5 pmol/10(6) PBMC after 1-2 h and could not be detected after 4 h in any of the three patients. Duplicate determinations deviated by less than 20%.