A high-performance liquid chromatographic assay was developed for the quantitative determination of hydrochlorothiazide (HCT) in human urine. Reversed-phase separation of HCT and the internal standard, trichloromethiazide (TCMT), was accomplished on a 300 X 3.9 mm mu Bondapak Phenyl column. Following solvent extraction, concentrations of HCT as low as 0.25 micrograms/ml in urine were quantified by UV detection at 280 nm. Detector response (peak-area ratio of HCT to TCMT) was linear to 50 micrograms/ml. No interferences were observed in the extracts obtained from drug-free urine nor from several antihypertensive agents which are commonly co-administered with HCT. This method has been routinely employed in bio-availability studies evaluating a variety of formulations as well as characterizing the pharmacokinetics of this drug from urinary excretion data.