A direct radioimmunoassay (RIA) for the quantitation of Retrovir (zidovudine, azidothymidine, AZT) in biological fluids has been developed. The assay is sensitive with an I50 value of about 30 nM and with a lower limit of detection of about 3 nM. Intra-assay precision gave sample coefficients of variation that ranged from 1.77 to 8.65% for the standard curve with human plasma. Inter-assay precision and accuracy were within acceptable limits. The RIA was validated by comparing results obtained form the analysis of rat plasma samples by both this RIA and a high-performance liquid chromatography method. None of the crossreactivities recorded should interfere with the assay system. The affinity constant of the antibody chosen for use was 1.4 z 10(9) L/mol.