A 3beta-hydroxy-Delta5-C27-steroid dehydrogenase active in bile acid biosynthesis was purified from pig liver microsomes by solubilization with sodium cholate and by chromatography on DEAE-Sepharose, aminohexyl-Sepharose, and blue Sepharose. The last step in the purification procedure was preparative isoelectric focusing in a Rotofor cell. The final enzyme preparation showed only one protein band upon SDS-polyacrylamide gel electrophoresis. The isoelectric point was estimated to about 7.0 and the apparent Mr was 36,000. The purified enzyme catalyzed the conversion of 7alpha-hydroxycholesterol, 7alpha,25-dihydroxycholesterol, 7alpha, 27-dihydroxycholesterol, and 3beta,7alpha-dihydroxy-5-cholestenoic acid into the corresponding 3-oxo-Delta4 compounds. The enzyme was inactive with C19 and C21 steroids as substrates. The enzyme was also inactive with C27 steroids having the 7-hydroxy group in beta- instead of alpha-position. The Km was found to be 0.30 and 0.32 microM with 7alpha-hydroxycholesterol and 7alpha, 27-dihydroxycholesterol as substrates, respectively. NAD+ was the preferred cofactor. A monoclonal antibody raised against the 3beta-hydroxy-delta5-C27-steroid dehydrogenase was prepared. After coupling to Sepharose, the antibody was able to bind the dehydrogenase and to decrease the conversion of 7alpha-hydroxycholesterol into 7alpha-hydroxy-4-cholest-3-one by more than 90%. The N-terminal amino acid sequence was determined and found to be similar but not identical with those of known 3beta-hydroxy-Delta5-steroid dehydrogenases active in steroid hormone biosynthesis. Thus, the purified enzyme active toward C27 steroids in bile acid biosynthesis appears to represent a novel type of 3beta-hydroxy-delta5-steroid dehydrogenase.