The concentrations of ACTH in the plasma and adenohypophysis and corticotrophin releasing factor (CRF) in the hypothalamus were determined and the functional capacities of the adenohypophysis and hypothalamus assessed in male rats treated with pentobarbitone and morphine. A single injection of morphine increased markedly the concentrations of ACTH in both the plasma and the adenohypophysis and the CRF content in the hypothalamus. When the opiate was given to rats injected 10 min previously with sodium pentobarbitone its effects were substantially reduced and the hypothalamo-pituitary-adrenocorticotrophic response to stress was abolished. The functional activity in vitro of pituitary tissue removed from control rats was not affected by the addition to the incubation medium of pentobarbitone and/or morphine. Pituitary glands of drug-treated rats also responded normally both in vivo and in vitro, with respect to ACTH secretion, to hypothalamic extracts prepared from control animals, CRF-41 and arginine vasopressin (AVP). On the other hand, hypothalami removed from pentobarbitone/morphine treated rats showed a marked reduction in their ability to secrete CRF in response to acetylcholine or 5-hydroxytryptamine as also did hypothalami from control animals after preincubation with pentobarbitone and/or morphine. The results suggest that the hypothalamus is an important site of action of pentobarbitone and morphine in modulating the functional activity of the hypothalamo-pituitary-adrenocortical system and are in accord with the view that the pentobarbitone/morphine-treated rat is suitable for the detection and quantification of potential corticotrophin releasing factors.