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To investigate the effects of sex hormones on 5-reductase, we examined 5-reductase mRNA content and enzyme activity in the adrenal cortex of peripubertal male and female rats. In male rats, the influence of castration or hormone-replacement treatment with dihydrotestosterone (5-DHT) on 5-reductase was assessed. To stimulate ovarian sex hormone production in immature female rats, the effect of a single injection of 5 IU pregnant mare serum gonadotrophin (PMSG) on 5-reductase was examined. The efficacy of the treatments was demonstrated by measuring serum LH and ventral prostate weight in male rats, and serum oestradiol and ovarian weight in female rats. Growth hormone was also measured across all treatments in male and female rats. Adrenal 5-reductase mRNA levels were determined by RNA blot analysis utilizing a rat 5-reductase cDNA as probe. 5-Reductase enzyme activity was estimated by isolating [ ³H]5-DHT by thin-layer chromatography after incubation with [³H]testosterone. The identity of the [³H]5-DHT formed was demonstrated by recrystallization of the derivatized DHT to constant specific activity. In controls, adrenal cortical 5-reductase mRNA content was nearly four times higher in immature female rats compared with intact peripubertal males. Castration resulted in a sevenfold increase in adrenal 5-reductase mRNA content compared with that in intact controls, while in DHT-injected castrated animals the mRNA level was nearly undetectable. The content of adrenal 5-reductase mRNA in anoestrous rats was nearly four times higher than in PMSG-treated animals. Adrenal 5-reductase activity was higher in immature female rats than in intact peripubertal males. Castrated rats displayed more than a threefold increase in 5-reductase activity over that of controls, whereas the activity values were below controls in castrated animals treated with DHT. In immature female rats treated with PMSG, 5-reductase activity decreased by 40% to that of anoestrous controls. These results indicate that in the rat adrenal cortex the content of mRNA encoding 5-reductase is negatively regulated by sex hormones presumably at the transcriptional level. Suppression of the enzymatic activity of adrenal 5-reductase by sex hormones is due to lower mRNA levels encoding this protein.