A sensitive method for the determination of 3α,5β-tetrahydroaldosterone (THALD) in human urine is described. The method uses high-performance liquid chromatography with chemiluminescence detection. Urinary THALD, released by enzyme hydrolysis, is isolated and concentrated using a Sephadex G-25M column and Bond-Elut C₁ cartridges, and then oxidized by copper(II) acetate to form the corresponding glyoxal derivative. The glyoxal derivative is converted into the chemiluminescent quinoxaline by reaction with 4,5-diaminophthalhydrazide. The chemiluminescent quinoxaline is separated within 50 min on a reversed-phase column (TSKgel ODS-120T) with isocratic elution, followed by chemiluminescence detection; the chemiluminescence is produced by the reaction of the quinoxaline with hydrogen peroxide in the presence of potassium hexacyanoferrate(III) in alkaline solution. The detection limit for THALD is 0.6 pmol (220pg) ml^–1 in urine [1.5 fmol (0.53 pg) per 20 µl injection] at a signal-to-noise ratio of 3. This method permits the sensitive and precise determination of THALD in human urine (50 µl) from normal subjects and a patient with primary aldosteronism.