BACKGROUND AND AIMS: Gastrointestinal smooth muscle is innervated bv non-adrenergic, non-cholinergic (NANC) inhibitory neurons. These inhibitory enteric neurons play an important role in gastrointestinal motility, and are involved in receptive and adaptive relaxation of the stotnach. 5-Hydroxytryptamine (5-HT) can exert multiple actions on the mamrnalian digestive tract, including contraction or relaxation of the stomach muscle. Nevertheless, the precise role of 5-HT in the control of gastrointestinal motility is not clear. The present study was designed to investigate the effects of 5-HT and related drugs on the NANC-mediated relaxation in the circular muscle of the rat gastric fundus and to characterize the receptor subtype irvolved. METHODS: We stimulated rat gastric fundus strips, contracted with 10 nM endothelin-1 (ET-1), by electric field stimulation method and then all results were recorded by Polygraph. RESULTS: 5-HT relaxed the rat gastric fundus strips precontracted with 10 nM ET-1 in a dose-dependent manner, which was affected by pretreatment with neither atropine (10 pM) nor guanethidine (50 pM). Furtheimore, the 5-HT, induced relaxation was partially blocked by pretreatment with tropisetron (a 5-HT, antagonist) but not by methiothepin (a 5-HT, antagonist) or ketanserin (a 5-HT, antagonist). The electrical field stimulation (EFS) caused frequency-dependent relaxation of fundus strips precontracted with 10 nM ET-1. The EFS response was not affected by pretreatment with atropine or guanethidine (100 pM, each), whereas tetrodotoxin (1 pM) completely abolished the response to EFS. The relaxation elicited by EFS was significantly reduced by N-nitro-L-arginine (I.-NNA) and N-nitro-L-arginine methyl ester (L-NAME), nitric oxide synthase inhibitors, and by tropisetron but not by methiothepin or ketanserin. The concentration-dependent relaxation to 5-HT was competetively antagonized by the 5-HT3 antagonist tropisetron, but not by methiothepin or kctanserin. CONCLUSIONS: Based on these results, it is concluded that stimulation of the 5-HT3 receptois results in relaxation and 5-HT is candidated as a mediator in NANC inhibitory neurotransmission in rat gasrtic fundus. Additionally, stimulation of neuronal 5-HT3 receptors results in an in!ibitory response mediated by the release of an unknown NANC neurotransmitter.