Neuromedin U-8 (NMU-8) is a peptide isolated from porcine spinal cord which contracts blood vessels and the uterus. Antisera were raised against NMU-8 and used in a radioimmunoassay (RIA) together with HPLC to characterize NMU-like immunoreactivity (NMU-LI) in tissues extracts of rat brain and gut and guinea pig gut. Samples of duodenum, ileum and distal colon were taken from both species, and processed for detection of NMU-LI by fluorescence immunohistochemistry. In RIA the antiserum had no cross-reactivity with neuropeptide Y, vasoactive intestinal peptide or the C-terminal hexapeptide of pancreatic polypeptide. Preincubation of antiserum with any of these peptides had no effect on the NMU-LI staining. In rats the highest content of NMU-LI was found in the ileum and the lowest in the cerebral cortex and striatum. HPLC studies showed that at least two molecular forms of NMU-LI were present in both species. In rat small intestine, subpopulations of submucous and myenteric neurones were stained; nerve fibres and terminals within these ganglia and in the mucosa were also seen. NMU-LI was sparse in the muscle. In guinea pig ileum small populations of nerve terminals were seen in both myenteric and submucous ganglionated plexuses. No endocrine cells were stained in either species.