A 1169 base pair fragment of bovine 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4 isomerase (3 beta-HSD) cDNA was used to quantitate 3 beta-HSD messenger RNA (mRNA) levels in the bovine ovary during the estrous cycle. The content of 3 beta-HSD protein was measured by immunoblot analysis using an antiserum developed in rabbits against human 3 beta-HSD, whereas 3 beta-HSD activity was measured using [3H]pregnenolone, [3H] dehydroepiandrosterone, and [3H]androst-5-ene-3 beta,17 beta-diol as substrates. There was a parallel increase in 3 beta-HSD mRNA, protein content, and enzymatic activity levels from days 1-3 after estrus to maximal values at 50-100% above control on days 8-11 after estrus. Thereafter, all values decreased progressively until days 16-17 before a dramatic fall to 5% or less than maximal values on days 18-20 after estrus. Almost superimposable results of enzymatic activity were obtained with the three substrates, thus suggesting a unique 3 beta-HSD or parallel changes in the activity of multiple 3 beta-HSDs. The above-described changes observed during the luteal phase are almost exclusively due to variations in corpora lutea. In fact, 3 beta-HSD activity in ovarian follicles was approximately 10,000 lower than that measured in corpora lutea. The close correlation observed over a wide range of 3 beta-HSD mRNA, protein content, and activity levels suggests that changes of ovarian 3 beta-HSD activity are controlled at the level of 3 beta-HSD gene expression and/or 3 beta-HSD mRNA stability.