Retrograde dye tracing was combined with immunohistochemistry to determine the distributions of nitric oxide synthase (NOS) immunoreactive nerve cells that project to prevertebral ganglia from the gastrointestinal tract and spinal cord of the guinea pig. An antiserum was raised against the neuronal form of NOS by selecting an amino-acid sequence specific to this form as immunogen. The antiserum recognised a single band at 150 kDa on Western blots of rat brain extract. Enteric nerve cells that were labelled by Fast Blue injected into the coeliac ganglion were not NOS immunoreactive in the small intestine, whereas 40-70% were reactive in the large intestine. Retrograde dye injected into the inferior mesenteric ganglion labels cells in the colon and rectum; 60-70% were immunoreactive for NOS. The NOS-immunoreactive nerve fibres arising in the intestine appear to end selectively around somatostatin-immunoreactive nerve cells in the coeliac and inferior mesenteric ganglia. Preganglionic nerve cell bodies in the intermediolateral column and dorsal commissural nucleus from T12 to L2 were labelled from the inferior mesenteric ganglion. Nearly 70% of neurons at each level were NOS immunoreactive. Thus, two sources of NOS terminals in prevertebral ganglia have been identified, intestinofugal neurons of the large, but not the small intestine, and sympathetic preganglionic neurons.