Female rats were ovariectomized and either sham-pinealectomized or pinealectomized on day 24 of age and exposed to a photoperiod of 12 h light: 12 h dark. In vivo and in vitro measures of adrenocortical function were made at 6, 7, and 8 weeks of age (18, 25, and 32 days post-pinealectomy, respectively). Pinealectomy diminished the post-castration rise in adrenal 5alpha-reductase activity in all age groups (P less than .05). The proportionate secretion of corticosterone (compared with the total steroid output) by adrenal slices was likewise enhanced (P less than .05) although the secretion and production of corticosterone in vitro was not altered. Pinealectomy substantially diminished (P less than .05) the in vivo secretion rates of reduced metabolites of corticosterone (dihydrocorticosterone and tetrahydrocorticosterone) without altering corticosterone secretion. Consequently, proportionate secretion of corticosterone in vivo was also enhanced (P less than .05). These findings are consistent with the notion that in ovariectomized rats removal of the pineal gland diminishes adrenal 5alpha-reductase activity without affecting ACTH secretion. However, in rats with ovaries intact, estrogen modified the effects of pinealectomy. Not only was the intra-adrenal metabolism of corticosterone diminished (higher proportionate output), but also resting levels of plasma corticosterone (P less than .01), corticosterone production in vitro (P less than .05), and total adrenal steroidogenesis in vitro (P less than .01) were increased. Thus, ACTH secretion may be enhanced following pineal removal in the presence of estrogen. The data suggest that the pineal gland, together with the ovaries, plays a role in the modulation of adrenal steroidogenesis.