1 A triple organ bath method allowing the synchronous recording of the motor activity of the circular muscle layer belonging to the oral and anal segments of guinea-pig small intestine adjacent to an electrically stimulated middle segment was developed to study the ascending and descending reflex motor responses. 2 Electrical field stimulation (0.8 ms, 40 V, 5 Hz, 10 s) applied to the middle part of the segments elicited tetrodotoxin (1 microm)-sensitive ascending and descending contractile responses of the nonstimulated parts, oral and anal, respectively. The ascending contraction was more pronounced as compared with the descending contraction. 3 In the presence of phentolamine (5 microm), propranolol (5 microm) and atropine (3 microm) a significant decrease in the amplitude of the ascending contraction was seen and a descending relaxation, instead of a contraction was observed. 4 Met-enkephalin applied at a single concentration (0.1 microm) or cumulatively (0.001-1 microm) inhibited both non-adrenergic non-cholinergic (NANC) descending relaxation and ascending contraction with similar efficacy but different potency, IC50 being 5.9 +/- 0.3 and 39.0 +/- 4 nm, respectively. Naloxone (0.5 microm) prevented the effects of Met-enkephalin. 5 L-NNA (0.5 mm), an inhibitor of nitric oxide synthesis, increased the ascending contraction and strongly reduced but not abolished the descending relaxation. l-Arginine (0.5 mm) restored the motor responses to the initial level in l-NNA-pretreated preparations, d-Arginine (0.5 nm) had no effects. 6 Met-enkephalin (0.1 microm) depressed the l-NNA-dependent increase of the ascending contraction and failed to change the l-NNA-resistant part of the descending relaxation. 7 Met-enkephalin did not alter spontaneous NANC mechanical activity. SNP (1 or 10 microm), an exogenous donor of nitric oxide, caused a concentration-dependent relaxation. The effects of SNP persisted in Met-enkephalin (0.1 microm)-pretreated preparations. 8 NANC reflex ascending contraction and descending relaxation were synchronously induced by a local nerve stimulation indicating a functional coactivation of NANC orally projected excitatory and anally directed inhibitory pathways. Acting prejunctionally, Met-enkephalin provided a negative controlling mechanism inhibiting both ascending and descending, mainly nitric oxide mediated, reflex responses. A higher sensitivity of the descending relaxation to Met-enkephalin was observed suggesting an essential role of opioid(s) in reducing the efficacy of descending motor activity.