In the steroidogenic pathways present in the gonads and adrenal cortex, 3β-hydroxysteroid dehydrogenase isomerase (3βHSD) is a key enzyme which controls the formation of Δ4-3-ketosteroids from Δ5-3β-hydroxysteroids. Herein, we used an antibody against human placental 3βHSD and a rat testicular 3βHSD cDNA probe to study the expression of rat liver 3βHSD mRNA and protein. Rat liver microsomal 3βHSD activity has been previously reported to exhibit a significant sex difference, with much higher activity in the male. We have shown an age-dependent increase in levels of immunoreactive 3βHSD through the time of maturation of the male rat. The immunoreactive protein, of similar molecular size to the human placental and rat testicular 3βHSD, was localized to the microsomal fraction of liver and was concentrated in pericentral locations. Immunoreactive protein was not detected in liver of immature (before 25 days of age) rats of either sex or in adult female liver. Northern blot analysis of liver and testicular RNA with a rat testicular 3βHSD cDNA probe revealed the presence of a 1.6-kilobase mRNA species in addition to the major 2.1-kilobase mRNA species in adult male liver, neither of which was detected in immature or adult female liver RNA. Hypophysectomy of female rats or treatment with testosterone implants caused induction of liver 3βHSD protein, while ontinuous infusion of GH to male rats decreased the level of 3βHSD protein. Similarly, the levels of the mRNA species were decreased after GH treatment. Using [3α-3H]dehydroepiandrosterone as substrate for 3βHSD activity, we determined the apparent Km for liver microsomal NAD+-dependent 3βHSD activity to be 20 μm in both adult male and female liver and was much greater than the Km of rat Leydig tumor 3βHSD activity (0.2 μm). Liver 3βHSD activity was inhibited by trilostane, a proven inhibitor of gonadal and adrenal 3βHSD activity. A rat liver 3βHSD cDNA was isolated from a male liver cDNA library that was closely related to the type II 3βHSD form of rat ovary but different from type III liver 3βHSD. The enzyme obtained upon expression of this cDNA had properties characteristic of male-specific NAD+-dependent liver microsomal 3βHSD (i.e. high apparent Km for dehydroepiandrosterone) and distinct from those of the high affinity gonadal type I 3βHSD. We conclude that liver 3βHSDs are distinct members of the 30HSD family of enzymes, and their male-specific expression is modulated via the sex-specific pattern of GH secretion.