The enzyme 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) is essential for the biosynthesis of all active steroid hormones. To date five distinct isoforms have been identified in the mouse. The different isoforms are indicated by roman numerals (I-V) in the chronological order in which they have been isolated. The different isoforms are expressed in a tissue- and developmentally specific manner and fall into two functionally distinct groups. 3 beta-HSD I, II, and III function as NAD(+)-dependent dehydrogenaselisomerases, and IV and V function as NADPH-dependent 3-keto steroid reductases. These latter two isoforms, therefore, are not involved in the biosynthesis of steroid hormones, but most likely in the inactivation of steroid hormones. In the adult mouse 3 beta-HSD I is expressed in the classical steroidogenic tissues, the adrenal glands and the gonads. 3 beta-HSD II and III are expressed in the liver and kidney, with III being the major isoform expressed in the adult liver. 3 beta-HSD IV is expressed almost exclusively in the kidney of both sexes, and expression of 3 beta-HSD V is observed only in the male liver starting late in puberty. In the fetal liver of both sexes, 3 beta-HSD I is the major or only isoform expressed at 13.5 days postconception and remains the major isoform until the day of birth, after which 3 beta-HSD III becomes the major isoform. Expression of 3 beta-HSD I in the liver decreases after birth and ceases by day 20 postnatally. Thus the liver expresses four distinct isoforms of 3 beta-HSD, I, II, III, and V, at different times during development. The mouse 3 beta-HSD genes, Hsd3b, have been mapped to a small region on mouse chromosome 3. Analysis of two yeast artificial chromosome (YAC) libraries identified one clone that contains the entire Hsd3b locus within a 1400-kb insert. Hybridization by Southern blot analysis of restriction-enzyme-digested YAC DNA using an 18-base oligonucleotide that hybridizes without mismatch to all known Hsd3b sequences indicates that there are a total of seven Hsd3b genes or pseudogenes in the mouse genome. Further analysis of mouse genomic DNA by pulse field gel electrophoresis suggests that all of the Hsd3b gene family is found within a 400-kb fragment.