Immunoreactive β-endorphin (ir-β-END) concentrations were measured in the hypophysial portal plasma of the male rat under urethane anesthesia. On the basis of immunochemical studies and gel fitration chromatography it appears that ir-β-END in rat hypophysial portal plasma is primarily β-endorphin (β-END) and not β-lipotropin (β-LPH). In addition, much of the ir-β-END in portal plasma may be of pituitary origin since acute hypophysectomy resulted in approximately an 80% decrease in the portal plasma concentration of ir-β-END. Nevertheless, in anesthetized animals that had been hypophysectomized acutely, portal plasma concentrations of ir-β-END were still 5 times those in systemic plasma, indicative of hypothalamic secretion of the peptide. The administration of morphine sulfate (3 mg/kg, i.v.) resulted in a decrease of ir-β-END concentrations from 3,157 ± 547 pg/ml to 1,044 ± 250 pg/ml. This effect was blocked by naltrexone (1 mg/kg, s.c.) pretreatment. Capsaicin (10 µg), which, when infused into the lateral cerebral ventricle of the rat, has been shown to decrease the amount of β-END in the hypothalamus, but not elsewhere in the central nervous system, selectively decreased the concentration of ir-β-END in portal plasma without changing systemic ir-β-END concentrations. These studies indicate that ir-β-END in portal plasma is probably β-END which is derived from neurons in the hypothalamus. Moreover, it is concluded that the regulation of the release of ir-β-END from these neurons involves opiate receptor mechanisms. The inhibitory influence of opiates on ir-β-END secretion may be indicative of a classical feedback regulation of ir-β-E