We report the isolation and characterization of a full length cDNA encoding rat 20 alpha hydroxysteroid dehydrogenase derived from rat corpus luteum RNA. The predicted amino acid sequence of the protein encoded by the 20 alpha HSD clone is composed of 323 amino acids possessing an approximate molecular weight of 37 kDa. The sequence of peptides derived from the purified protein was found in the translated sequence of the open reading frame. cDNA and amino acid sequence indicate that the rat ovarian 20 alpha HSD belongs to the aldo-keto reductase family of enzymes. Northern analysis revealed a 1.2 Kb 20 alpha HSD mRNA in corpora lutea undergoing luteolysis. Prolactin reduced markedly 20 alpha HSD mRNA expression. No signal was detected in other tissues examined, demonstrating the specific expression of this enzyme in the corpus luteum.