The role of nitric oxide (NO) in 5-HT-induced non-adrenergic non-cholinergic (NANC) relaxations was studied on circular muscle strips of the canine ileocolonic junction (ICJ) and terminal ileum. During an acetylcholine-induced contraction, NO (10(-5) M) evoked a transient relaxation, whereas 5-HT (10(-4) M) caused an initial NANC relaxation followed by a contraction. This initial relaxation to 5-HT, but not the relaxation to NO, was significantly inhibited by the stereospecific inhibitors of the NO biosynthesis NG-monomethyl-L-arginine (L-NMMA) and NG-nitro-L-arginine (L-NNA). L-arginine, but not D-arginine, prevented the inhibitory effect of L-NMMA and L-NNA. The enantiomer of L-NMMA, D-NMMA, had no effect. Hemoglobin abolished the NO-induced relaxations and significantly inhibited the relaxation to 5-HT. From these experiments it is concluded that the 5-HT-induced NANC relaxation is mediated by NO or a NO releasing substance.