The enzyme 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4 isomerase (3 beta-HSD) catalyzes the obligatory oxidation and isomerization of delta 5-3 beta-hydroxysteroid precursors into delta 4-3-ketosteroids which lead to the formation of all classes of steroid hormones. We report the molecular cloning of a third type of cDNA clone encoding rat 3 beta- HSD isolated from a rat liver lambda gt11 cDNA library. The nucleotide sequence of 1955 bp determined from overlapping cDNA clones predicts a protein of 372 amino acids which displays 80% similarity with that of rat type I and type II 3 beta-HSD proteins. RNA blot analysis reveals the presence of mRNA transcripts of 2.1 kb in male liver in contrast to the 1.7 kb mRNA species detected in adrenal and gonad poly(A)+ RNA. Ribonuclease protection assays using type I, type II and type III specific cRNA probes demonstrate the liver-specific expression of type III mRNA while the two others are expressed in adrenals and gonads. The type III mRNA species was below the detection limit in intact female liver while in hypophysectomized females, its accumulation was restored to 55% of the levels measured in intact or hypophysectomized male rats. The present data describe the presence of a third type of 3 beta-HSD mRNA species and its marked sexual dimorphic gene expression in the liver which apparently results from pituitary hormone-induced gene repression in female rat liver tissue.