The distribution of the enzyme Δ5-3β-hydroxysteroid dehydrogenase (3βHSD) was studied histochemically in sections of ovaries from normal golden hamsters. During the estrous cycle, the enzyme activity increased in new corpora lutea to a maximum at 2 days after ovulation and then declined. The theca of growing follicles gradually developed activity during the cycle, attaining maximal activity on Day 4 before ovulation when corpora lutea were inactive. Only on Day 4 of the cycle did the granulosa show 3βHSD. The interstitium was histochemically active at all times. The corpora lutea of pregnancy developed strong enzyme activity by Day 2 which was maintained until Day 15 and faded before delivery on Day 16. Growing follicles during pregnancy had activity in the theca on all days, but it became weaker on Day 16. The granulosa of follicles showed some 3βHSD by Day 4 of pregnancy, and this increased after Day 8 to become as strong as that in corpora lutea on Day 12. The strong 3βHSD activity in the granulosa persisted until after delivery. Follicles did not ovulate before or after delivery, and the corpora lutea and antral follicles of pregnancy disappeared during the first few days of lactation. By 8 days of lactation, only interstitium had 3βHSD. We propose that the changes in the distribution and activity of 3βHSD in the ovary of the golden hamster reflect the changing ratios of gonadotropins and the resulting effects on ovarian steroid secretion.