The effects of gonadectomy and of the administration of gonadal steroids on the content of substance P in the anterior pituitary (AP-SP) were studied in adult rats. The effect of gonadal status on the AP-SP content of thyroidectomized (TX) rats was also studied. We have confirmed that the AP-SP content in adult males is higher than that in adult females. Ovariectomy (OVX) caused an increase in AP-SP content which was apparent 6 days after surgery. Estradiol (E2; 2 micrograms/rat daily) administered for 13 days beginning the day after OVX prevented the increase in AP-SP content induced by OVX. Orchiectomy of adult rats had no effect on AP-SP content 14 and 45 days after surgery. E2 administered to adult female rats for 13 days caused a reduction in the AP-SP content, whereas dihydrotestosterone (0.2 mg/rat daily for 13 days) caused an increase that was even more pronounced in TX rats. E2 administration to TX adult female rats caused a significant decrease in the AP-SP content both when treatment was begun on the day after surgery or 2 weeks later. Administration of T4 (1.5 and 25 micrograms/100 g BW daily for 7 days) to rats made hypothyroid by thyroidectomy 2 weeks earlier abolished the increase in AP-SP content seen in TX animals. Neither E2 nor dihydrotestosterone had an effect on the substance P content of any of the brain regions examined. The AP-SP content of pregnant or lactating rats was not different from that of age-matched controls. The content of substance P in the AP and median eminence did not vary significantly throughout the estrous cycle. The data indicate that AP-SP content is dependent on the gonadal status of the animal and that gonadal steroids interact with thyroid hormones in the regulation of substance P turnover in the AP.