Several lines of experimental evidence suggest that acetylcholine (ACh) is excitatory to the hypothalamic-pituitary-adrenal (HPA) axis. Since previous experiments have shown that ACh does not affect pituitary adrenocorticotropin secretion in vitro, we hypothesized that ACh stimulates the HPA axis by causing hypothalamic corticotropin-releasing hormone (CRH) secretion. We examined this hypothesis using an organ culture system that measures the ability of single rat hypothalami to secrete immunoreactive CRH (IR-rCRH) in vitro. ACh stimulated hypothalamic IR-rCRH secretion in a dose-dependent fashion, at concentrations ranging from 3.3 × 10–10 to 10–5M. This effect was antagonized by the simultaneous presence of atropine and hexamethonium, a muscarinic and a nicotinic receptor antagonist, respectively (p < 0.05). Further evidence for the cholinergic regulation of the CRH neuron was provided by the findings that both carbachol, a muscarinic receptor agonist, and nicotine, a nicotinic receptor agonist, stimulated IR-rCRH secretion in a dose-dependent fashion. These effects were antagonized by atropine and hexamethonium, respectively, suggesting that both muscarinic and nicotinic receptors are involved in the process. ACh stimulated hypothalamic IR-rCRH secretion in the presence of phentolamine, an α-adrenergic antagonist, and ritanserin, a serotonin receptor antagonist, suggesting that the cholinergic stimulation of CRH secretion is not mediated by α-adrenergic or serotonergic interneurons. We conclude that ACh stimulates hypothalamic CRH secretion via both muscarinic and nicotinic receptor mechanisms. This effect is not mediated by a serotonergic or α-adrenergic interneuron. These data suggest that ACh may be implicated in the regulation and the stress activation of the HPA axis