In previous research we found that interleukin-2 (IL-2)-induced corticotropin-releasing hormone (CRH) release in vitro is mediated by cholinergic activation of nitric oxidergic (NOergic) neurons. The NOergic neurons release nitric oxide that stimulates CRH release. To further characterize the mechanism of IL-2-induced CRH release, the possible role of nicotinic as well as muscarinic receptors in IL-2-stimulated CRH release was evaluated. Medial hypothalamic (MH) explants from adult male rats were preincubated in Krebs-Ringer (KRB) buffer for 45 min followed by incubation for an additional 30 min in fresh KRB or KRB containing various compounds. As previously reported, acetylcholine (ACH) stimulated CRH release in a dose-related fashion. IL-2 (10(-13) M) stimulation of CRH release was unaffected by the lower concentration of ACH (10(-9) M), but surprisingly was inhibited by a 100-fold higher concentration. Atropine (ATR) (10(-7) M) blocked CRH release induced by ACH (10(-7) M) and the release of CRH induced by IL-2. The cholinergic agonist carbachol (CAR) (10(-7) M) also released CRH and this action was blocked by ATR (10(-7) M). CRH release in the presence of CAR was lowered below basal when the concentration of ATR was increased to 10(-6) M. In contrast to ACH, CAR had an additive effect to release CRH when combined with IL-2 (10(-13) M). Nicotine (10(-7) M) also stimulated CRH release and this stimulation was completely blocked by 10(-6) M but not by 10(-7) M of the nicotinic receptor blocker, hexamethonium (HEX). The lower concentration of HEX blocked the stimulatory effect of ACH (10(-7) M) and IL-2 on CRH release. Combined blockade with ATR plus HEX completely blocked the action of ACH and even reduced the CRH concentration to below basal values. Furthermore, combined blockade completely blocked the release of CRH induced by IL-2. We conclude that nicotinic as well as muscarinic receptors play an important role in CRH release, and that they both act to mediate IL-2-stimulated CRH release.