A rapid, sensitive and reproducible reversed-phase high-performance liquid chromatographic assay was developed for the determination of norfloxacin. Following protein precipitation with 10% trichloroacetic acid, norfloxacin and the internal standard enoxacin were extracted from plasma with chloroform, dried and reconstituted in the mobile phase. The chromatographic separation of norfloxacin and the internal standard enoxacin was achieved on a C8 column with fluorescence detection set at 280 and 418 nm for excitation and emission, respectively. The peaks with a resolution factor greater than 1.5 were free from interferences. Excellent linearity (r2 > or = 0.998) was observed over the concentration range 0.025-5.0 micrograms/ml in plasma. The inter-assay variability was 13.6% or less at all concentrations examined. The suitability of the assay for pharmacokinetic studies was determined by measuring norfloxacin concentration in rat plasma after administration of a single intravenous 10 mg/kg dose.