We have previously purified the 20α-hydroxysteroid dehydrogenase (20α-HSD) molecule from normal mature rat ovarian cytosol [Biochim. Biophys. Acta, 1991, 1079: 112-118]. In order to further characterize the 20α-HSD and to obtain information for molecular cloning of the enzyme, we determined its N-terminal amino acid sequence. The sequence was shown to be Ser-Lys-lle-Gln-LysMet-Glu-Leu-Asn-Asp-Gly-His-Ser-Ile-Pro-Val-Leu-Gly-Phe-Xaa-Thr. A search of the SWISS-PROT protein sequence database for N-terminal amino acid sequence similarities revealed 6 highly homologous proteins, including bovine prostaglandin F synthase, rat liver 3α-hydroxysteroid dehydrogenase, bovine lens aldose reductase, human aldose reductase, frog ε-crystallin and human liver chlordecone reductase, suggesting that rat ovarian 20α-HSD belongs to the aldo-keto reductase gene superfamily.