Alcohol (EtOH) treatment increases plasma ACTH levels in the rat, a response thought to depend at least partially on endogenous corticotropin-releasing factor (CRF). However, the inability of CRF antibodies to completely abolish EtOH-induced activation of the pituitary has suggested the possible importance of other factors. Because vasopressin (VP) may be acutely released by alcohol in the rat, and because this peptide interacts with CRF to promote ACTH secretion, we investigated its role in the hypothalamic-pituitary (HP) axis' response to one acute injection of EtOH (3 g/kg, i.p.). As expected, blockade of pituitary CRF receptors significantly blunted EtOH-induced ACTH secretion. Administration of a VP antagonist also interfered with this response, though to a lesser extent, while concomitant administration of both CRF and VP antagonists totally abolished it. These results indicate that both CRF and VP participate in the ability of alcohol to stimulate corticotrophs' function. Another hypothesis still awaiting confirmation is whether alcohol stimulates the activity of hypothalamic neurons important for ACTH release, which are primarily found in the paraventricular nucleus (PVN) of the hypothalamus. We therefore determined whether EtOH upregulated transcripts of the immediate early gene (IEG) NGFI-B, as well as those of CRF and VP. In the parvicellular portion of the PVN (pPVN), NGFI-B mRNA and CRF heteronuclear (hn) RNA levels increased within 20 min of EtOH treatment. Despite this robust response, densitometric assessments of CRF mRNA levels failed to indicate a significant elevation over control levels at any of the time points studied, a phenomenon that might be at least in part due to the abundance of these transcripts under basal conditions. Constitutive levels of VP hnRNA levels were very low in the pPVN, but high in the magnocellular division (mPVN). These signals tended to be increased in rats injected with alcohol, but these responses did not reach statistical significance. However, the mRNA encoding VP became significantly more expressed 3 h post-alcohol exposure, suggesting that gene expression had increased. Collectively, these results indicate that alcohol stimulates the activity of neurons in both the parvo- and the magnocellular portions of the PVN, though transcriptional activation of the CRF and VP genes in the PVN exhibit distinct time courses. Whether the influence of the drug is direct or is exerted through afferents to the PVN, and whether increased hypothalamic neuronal activity plays a role in mediating the acute ACTH response to alcohol, are issues that have not yet been adequately resolved.