A fluorescence polarization immunoassay (FPIA) procedure for measuring isepamicin, a new aminoglycoside antibiotic, was evaluated. The range of the assay studied was from 0.3-50 micrograms/ml and the calibration curve can be stored for at least 32 days. There was no interference from hemoglobin (up to 400 mg/dl) and bilirubin (up to 20 mg/dl). Within-run precision on each 20 replicates at 5, 15, and 25 micrograms/ml yielded a coefficient of variation (CV) of 1.44-2.52%; between-run precision on analyses at 5, 15, and 25 micrograms/ml over five periods yielded a CV of 1.26-1.59%. Cross-reactivities of the assay system with tobramycin and amikacin were null, but that with gentamicin, of which isepamicin is a derivative, was approximately 14-29%. Data obtained by microbiological assay and FPIA and by high-performance liquid chromatography and FPIA correlated significantly, with coefficients of correlation being 0.980 (n = 70) and 0.965 (n = 39), respectively. The FPIA offers a rapid, efficient, and accurate system for therapeutic monitoring of isepamicin plasma levels.