An automated fluorescence polarization immunoassay for the determination of vancomycin levels in serum was evaluated. The vancomycin assay is a homogeneous competitive inhibition immunoassay based on changes in fluorescence polarization that occur with antibody binding. This assay was compared with a liquid chromatographic assay and an agar well diffusion bioassay method by using clinical serum specimens and controls. Linear regression analysis of the data obtained on clinical specimens by the three methods resulted in correlation coefficients of 0.97 for the fluorescence polarization immunoassay versus the liquid chromatographic assay (n = 60), 0.90 for the fluorescence polarization immunoassay versus the bioassay (n = 57), and 0.90 for the liquid chromatographic assay versus the bioassay (n = 57). Repetitive analysis of control sera containing 7, 35, and 75 micrograms of vancomycin per ml by the fluorescence polarization immunoassay yielded coefficients of variation of 3.0, 1.7, and 2.3, respectively. No interference was demonstrated in spiked hemolytic, lipemic, or icteric sera, and the assay was free of matrix effects. The automated fluorescence polarization immunoassay system offers a rapid, efficient, and accurate method for monitoring vancomycin serum levels for both toxicity and efficacy.