The mechanisms of neurogenic relaxation in the longitudinal muscle of the isolated canine colon and its modification by enteric substances were investigated. Relaxations induced by transmural electrical stimulation with electrical pulses, nicotine or K+ in the muscle strips contracted with bradykinin and treated with atropine were attenuated but not abolished by NG-nitro-L-arginine (L-NA), and the inhibition was reversed by L-arginine. Oxyhemoglobin and ouabain inhibited the response, whereas K+ channel inhibitors, such as glibenclamide, tetraethylammonium, apamin and charybdotoxin, were without effect. In L-NA-treated strips, stimulation-induced relaxations were reduced by ouabain but not by oxyhemoglobin. Among substances tested, only norepinephrine, ATP, vasoactive intestinal peptide (VIP) and galanin produced relaxations. However, alpha- and beta-adrenoceptor antagonists and aminophylline did not alter the response to nerve stimulation. In the strips made unresponsive to VIP and galanin, stimulation-induced relaxations were not influenced. Indomethacin, calcitonin gene-related peptide, cholecystokinin, peptide YY, substance P and serotonin did not modulate the neurogenic response. It is concluded that the relaxation associated with nerve stimulation is mediated by nitric oxide (NO) synthesized from L-arginine and also by substance(s) activating the electrogenic Na+ pump but not that opening K+ channels. Norepinephrine, ATP, VIP and galanin can be excluded as candidate inhibitory neurotransmitters, and the substances used so far are unlikely to modulate inhibitory nerve function.