Nitric oxide has been implicated as the neurotransmitter mediating internal anal sphincter (IAS) relaxation during the rectoanal inhibitory reflex. However, there has been no direct demonstration of a nitrergic rectoanal neuronal pathway appropriate to mediating the reflex. This study combined retrograde neuronal tracing techniques with enzyme histochemistry in a guinea-pig model. Wheatgerm agglutinin conjugated to horseradish peroxidase was injected into the IAS. Transported tracer was demonstrated in neurones of the myenteric ganglia of the distal rectum and all labelled neurones showed co-localization with nitric oxide synthase as revealed by reduced nicotinamide adenine dinucleotide phosphate diaphorase histochemistry. In vivo anal canal manometry showed that the mean maximal resting pressure was 16 (8-20) cmH2O and confirmed the presence of the rectoanal inhibitory reflex. In vitro organ bath studies showed that strips of IAS developed spontaneous myogenic tone and relaxed in response to intrinsic nerve stimulation. Addition of N omega-nitro-L-arginine (L-NOArg) reduced the relaxant response in a dose-dependent fashion; the relaxant response was maximally reduced by a mean(s.e.m.) 35.2(3.8) per cent (P < 0.001) at a concentration of 3 x 10(-5) mol/l L-NOArg. This study provides direct anatomical evidence of a descending nitrergic rectoanal neuronal pathway in a guinea-pig model. In vivo anal manometry and in vitro organ bath studies provide additional evidence that this pathway is responsible for the inhibitory motor innervation of the rectoanal inhibitory reflex.