A simple, precise and sensitive method for separation and determination of total bile acid sulfates in human urine is described. The sulfate fraction of urinary bile acids was separated with lipophilic anion exchange gel, piperidinohydroxypropyl Sephadex LH-20 after sample clean-up with Sep-Pak C18 cartridge. The obtained sulfate fraction was submitted to solvolysis with a small volume of dimethoxypropane-HCl solution and subjected to enzymatic-fluorimetrical assay using 3 alpha-hydroxysteroid dehydrogenase and resazurin. In this method, no influence of existing salts in the reaction mixture on fluorescence intensity was observed and solvolysis reaction was almost complete. Overall recoveries of glycine- and taurine-conjugated bile acid 3-sulfates from normal urine ranged from 90.5 to 93.7% and those of unconjugates from 48.7 to 78.0%. The sensitivity of the described method enabled to estimate total bile acid sulfates with 0.5 ml of normal urine and precision tests showed the satisfactory accuracy. The level of total urinary bile acid sulfates was estimated on some patients with hepatobiliary diseases and healthy subjects.