Two solid phase, nonradioactive immunoassays were developed and evaluated for the determination of zidovudine (Retrovir, ZDV, AZT) concentrations in serum. The first, an enzyme-liked immunosorbent assay (ELISA), used an anti-AZT monoclonal antibody (MAb) and subsequent alkaline phosphatase second antibody system for the detection method. The second, a time-resolved fluoroimmunoassay (TR-FIA), used a polyclonal anti-AZT antibody followed by a europium-labeled goat anti-rabbit immunoglobulin (IgG) as the fluorescent probe. The ELISA had a detection range from 125 to 4,000 nMand a 50% inhibitory concentration (IC50) of about 500 nM. Development of the TR-FIA was based on a very sensitive detection system of metal chelate chemistry and time-resolved fluorometry. The standard curve in the TR-FIA was from 5 to 4,000 nM with an IC50of 200 nM. Intra- and interassay precision of the ELISA was good, with coefficients of variation from 3.9 to 7.3% and 3 to 17%, respectively, while the same values for the TR-FIA were 6.2 to 10.9% and 5.4 to 19.9%, respectively. The results obtained from each method were compared individually with those of high-performance liquid chromatography (HPLC) and radioimmunoassay (RIA). There was good agreement among the results obtained by each method. These two methods enable laboratories not licensed for radioisotopes to analyze potentially infectious samples without aerosol-forming centrifugation.