Abstract The cytoplasm of rat kidney contains a 17β-hydroxysteroid dehydrogenase which has been purified 12-fold by precipitation of the 20 000 × g supernatant with ammonium sulfate. The enzyme reacts with both phenolic and neutral steroids; either NAD or NADP can be utilised as cofactors. The oxidation of testosterone to androst-4-en-3, 17-dione shows a maximum at pH 8.7, whereas the reduction of androst-4-en-3, 17-dione to testosterone is optimum at pH 6.6. The Michaelis-Menten constants were found to be 18.5 · 10 −5 M for testosterone and 40 · 10 −5 M for NAD. A value of 9.1 · 10 −9 M was obtained for the equilibrium constant. The enzyme shows an activation energy of 13.2 kcal/mole and an inactivation energy of −24.0 kcal/mole within the range of 10–37°; the Q 10 values varied between 1.6 and 1.5. The enzymic activity is inhibited by SH blocking agents, but activated by zink ions in low concentrations (10 −5 M). The physiological significance of the 17β-hydroxysteroid hydroxysteroid dehydrogenase of rat kidney is discussed briefly.