7β-Hydroxysteroid dehydrogenase (7β-HSD), a specific enzyme active in the metabolization of 7β-hydroxycholesterol, was purified about 300-fold from male rabbit liver microsomes using ion exchange, hydroxylapatite, 2'5'ADP Sepharose 4B, and high-performance liquid chromatography on the basis of its catalytic activity.The specific activity of the purified enzyme was 276nmol/min/mg protein. The molecular weight of the purified enzyme was 34,000. The preferred coenzyme was β-NADP + . The optimum pH for oxidation was around 7.7 in potassium phosphate buffer, and 11.0 in glycine-NaOH buffer. The purified enzyme catalyzed the synthesis of not only 7β-hydroxycholesterol but also corticosterone and hydrocortisone. Enzyme activities toward these three substrates accompanied all purification steps of 7β-HSD. The amino acid sequence of the N-terminal of the purified enzyme showed that 7β-HSD had sequence similarity to rabbit type I 11β-hydroxysteroid dehydrogenase (11β-HSD), indicating that 7β-HSD may belong to the rabbit type I 11β-HSD family and may play the same role in the metabolism of 11-hydroxysteroids and 7-hydroxysterols.