Intensive research into the biological roles of nitric oxide has shown that this tiny molecule is of vital physiological significance in numerous organ systems including the gastrointestinal tract, where nitric oxide has been proposed as an inhibitory enteric neurotransmitter. This paper outlines experiments using retrograde neuronal tracing and enzyme histochemistry in a guinea-pig model which provided the first direct anatomical evidence of a descending nitrergic rectoanal neuronal pathway appropriate to mediating relaxation of the internal anal sphincter during the rectoanal inhibitory reflex. Studies of human tissue showed that the in vitro responses of isolated strips of human rectum were typical of non-sphincter specialized gastrointestinal smooth muscle, that nitric oxide is involved in neurogenic relaxation of the rectum and that nitric oxide synthase immunocytochemistry identified a subpopulation of neurones in the myenteric ganglia and immunoreactive profiles within both layers of the muscularis propria of human rectum. Taken together, these data provide pharmacological and anatomical support for the hypothesis that nitric oxide acts as a functionally important mediator in the innervation of human anorectum.