The enzyme 3β-hydroxysteroid dehydrogenase (3βHSD) is essential for the biosynthesis of all steroid hormones. To date this laboratory has isolated and characterized five distinct 3βHSD cDNAs in the mouse (3βHSD I-V). The different isoforms fall into two functionally distinct groups. 3βHSD I and III function as dehydrogenase/isomerases and 3βHSD IV and V function as 3-ketosteroid reductases. Previously it was shown that the liver of the adult mouse expresses 3βHSD II, III and V, with 3βHSD III being the major isoform. This study examines the expression of the different forms of 3βHSD mRNAs and proteins in the livers of male and female mice during fetal and postnatal development. 3βHSD I, which in the adult mouse is expressed only in the gonads and adrenal glands, is the major isoform expressed in both male and female livers during fetal development until the first postnatal (pn) day after which time 3βHSD III becomes the major isoform. Expression of 3βHSD I mRNA and protein completely ceases after day 20 pn. The expression of 3βHSD V is first detected at day 40 pn and is observed only in the male. Very low expression of 3βHSD II mRNA is detected throughout development. Previous characterization of enzymatic activity of the expressed proteins showed that 3βHSD I exhibits lower K m values for the Δ 5 -3β-hydroxysteroids than 3βHSD III, indicating that 3βHSD I functions as a more efficient 3β-hydroxysteroid dehydrogenase/isomerase than 3βHSD III. The results of this study suggest that the liver may play an important role in the biosynthesis of steroid hormones during murine fetal development.