1. The role of nitric oxide (NO) in non-adrenergic non-cholinergic (NANC) neurotransmission was studied on circular muscle strips of the canine lower oesophageal sphincter (LOS). Electrical field stimulation evoked frequency-dependent relaxations, which were resistant to adrenergic and cholinergic blockade and abolished by tetrodotoxin. 2. Exogenous administration of NO induced concentration-dependent and tetrodotoxin-resistant relaxations which mimicked those in response to electrical stimulation. 3. NG-nitro-L-arginine (L-NNA), a stereospecific inhibitor of NO-biosynthesis, inhibited the relaxations induced by electrical stimulation but not those by exogenous NO or vasoactive intestinal polypeptide (VIP). 4. The effect of L-NNA was prevented by L-arginine, the precursor of the NO biosynthesis but not by its enantiomer D-arginine. 5. Haemoglobin abolished the NO-induced responses and reduced those evoked by electrical stimulation. 6. Cumulative administration of VIP induced concentration-dependent relaxations, which were slow in onset and sustained. A complete relaxation to VIP was not achieved and the relaxations were not affected by L-NNA. 7. In conclusion, our results provide evidence that NANC relaxations are mediated by NO, suggesting NO or a NO releasing substance as the final inhibitory NANC neurotransmitter in the canine LOS.