An enzymatic determination of urinary total 17β-hydroxysteroids is described. The principle of the method is as follows: after hydrolysis with β-glucuronidase and solvolysis, the ethyl acetate layer, which is washed with alkaline solution and water, is transferred to two test tubes, then each of them is evaporated. To one test tube is added 3β,17β-hydroxysteroid dehydrogenase (EC 1.1.1.51, from P. testosteroni), NAD+, 2-ρ-iodophenyl-3-ρ-nitrophenyl-5-phenyltetrazolium chloride (INT), and diaphorase (EC 1.6.99.2, from C. kluyveri) for determination of total 3β,17β-hydroxysteroids. To another test tube is added 3β-hydroxysteroid oxidase (EC 1.1.3.6, from B. sterolicum) for determination of total 3β-hydroxysteroids. The value of 17β-hydroxysteroids is calculated by subtracting the value for 3β-hydroxysteroids from the value for the 3β,17β-hydroxysteroids.