We investigated the distribution pattern of nitric oxide (NO) synthesizing nerve cell bodies and axons in the biliary system of the guinea-pig using immunohistochemistry for nitric oxide synthase (NOS). Nerve fibres staining for NOS were found to contact non-vascular smooth myocytes and to course beneath the epithelium. No perivascular NOS fibres were observed. The innervation density varied in different parts of the biliary tree. The lower portion of the common bile duct was more richly innervated than the remaining parts of the duct system. NOS-containing neurones encompassed a subpopulation of intramural ganglion cells. Sympathetic neurones in the coeliac ganglion were not stained. It is suggested that intrinsic NOergic neurones are involved in inhibitory motor control of the biliary musculature, including the sphincter of Oddi.