Previous studies using the isolated rat hypothalamus in vitro have shown that both acetylcholine and 5-hydroxytryptamine (5-HT) stimulate the secretion of bioactive corticotrophin-releasing factor (CRF). However, the CRF complex consists of a number of bioactive substances, and in this study we examined the effect of acetylcholine and 5-HT on the release of immunoreactive (ir)-CRF-41 and ir-arginine vasopressin (AVP) in vitro. Acetylcholine caused a dose-dependent (10 pmol-10 nmol/l) release of both neuropeptides, and the effect was partially antagonized by both atropine and hexamethonium. Nicotine (0.1-10 μmol/l) also stimulated the release of both peptides, whereas bethanacol had no effect on AVP release, but had a variable action on CRF-41 release. 5-HT caused a dose-dependent (10 pmol-1 nmol/l) stimulation of CRF-41 release without any effect of AVP release, and this effect was antagonized by cyproheptadine, suggesting the participation of specific 5-HT receptors.