Alcohol (EtOH) releases ACTH through mechanisms that ultimately depend on the presence of endogenous corticotropin-releasing factor (CRF), but we still do not know where alcohol acts within the hypothalamic-pituitary-adrenal (HPA) axis. The present study was designed to determine the respective importance of the activation of pituitary and/or hypothalamic CRF receptors in mediating the stimulatory effect of EtOH on ACTH secretion. We used two CRF antagonists: (1) alpha-helical CRF9-41 (alpha-hel ant.), which is very effective in interfering with biological responses mediated by brain CRF receptors, but relatively impotent in blocking pituitary CRF receptors; and (2) astressin, {cyclo(30-33)[DPhe12,Nle21,38,Glu30,Lys33]r/h CRF12.41}, a member of the newest generation of antagonists capable of significantly blocking both brain and pituitary CRF receptors. The role of pituitary CRF receptors was shown by the ability of i.v. injected astressin, at doses shown to completely block CRF-induced ACTH release, to significantly (P < 0.01) reduce the ACTH response to EtOH (1.5 or 3 g/kg, i.p.). The importance of hypothalamic CRF receptors was tested by injecting alpha-hel ant. intracerebroventricularly (i.c.v.). We reasoned that if this antagonist leaked to the pituitary following administration into the ventricle, it would not be able to act directly on the corticotrophs. The ACTH response to EtOH was modestly and not significantly (P > 0.05) blunted by i.c.v. alpha-hel ant. Similarly, the stimulatory effect of alcohol on hypothalamic neuronal activation, measured by increases in the immediate early gene NGFI-B mRNA levels, was only slightly altered by blockade of hypothalamic CRF receptors. Collectively, these results suggest that during acute alcohol treatment, few brain pathways mediating the stimulatory effect of EtOH on ACTH release depend on the activation of CRF synapses. The induction of immediate early gene transcripts by alcohol similarly relies on hypothalamic circuits that stimulate CRF neurons relatively independently of the activation of CRF receptors. In contrast, functional pituitary CRF receptors are essential for the ACTH response to the drug.