The mechanisms of regulation of ovarian 7,12-dimethylbenz[a]anthracene (DMBA) hydroxylase were investigated with respect to hormonal requirements and effects of the antiestrogen tamoxifen and known inducers of cytochrome P-450. The DMBA hydroxylase is increased endogenously about 3-fold in the proestrus phase as compared to the metestrus/diestrus phases (M. Bengtsson and J. Rydstrom, Science, 219 (1983) 1437-1438). A similar effect was caused by the gonadotropins follicle-stimulating hormone (FSH) and luteinizing hormone (LH) whereas pregnant mare's serum gonadotropin (PMSG) brought about a 3-7-fold increase, suggesting that the estrus cycle-dependence of the DMBA hydroxylase was due directly or indirectly to gonadotropins. In contrast, differentiation of granulosa/theca cells to corpus luteum cells after ovulation, caused by administration of human chorionic gonadotropin (hCG), led to a marked decrease in activity. The activity was not specific for DMBA since substitution of this hydrocarbon for benzo[a]pyrene (BP) as substrate gave similar results. A possible role of estrogens in this context was investigated by the administration of tamoxifen simultaneous with gonadotropin treatment, which caused a partial inhibition of the hydroxylase activity. Both estradiol and 3-methyl-cholanthrene (MC) increased DMBA hydroxylase but the effects of these agents were not additive. In contrast, the effects of estradiol and MC were partially additive to that of gonadotropin. On the basis of these results, it is proposed that the rat ovary contains one or several aryl hydrocarbon hydroxylases located in the granulosa/theca cells which are regulated by estrogens, MC and beta-naphthoflavone (BNF) and that the role of gonadotropins is to proliferate granulosa/theca cells.