The quantitative distribution of neuropeptide Y (NPY) immunoreactivity has been determined along the length of the gastrointestinal tract in three mammalian species; rat, pig, and guinea pig. The peptide was shown to be present in all regions studied and in all three species. Exceptionally high concentrations were found in the region of the lower esophageal sphincter. Pretreatment of rats with 6-hydroxydopamine depleted NPY concentrations by 30-40%, indicating that NPY is colocalized in part with adrenergic nerves. Characterization of the NPY immunoreactivity by high-pressure liquid chromatography revealed a single major peak. NPY immunoreactivity derived from rat extracts eluted consistently earlier from the column than synthetic porcine standard, indicating minor species differences. Pharmacological studies using longitudinal muscle from guinea pig terminal ileum demonstrated that NPY caused a dose-dependent inhibition of the electrically stimulated, neurally mediated contraction of longitudinal smooth muscle. This suggested that NPY may act presynaptically to inhibit cholinergic transmission. The effects of various NPY fragments were also tested on the same preparation. The C-terminal fragments were active but were considerably less potent than NPY, while the free acid form of NPY and N-terminal fragment (1-19) were completely inactive. Thus, this study has demonstrated the presence of NPY in the gastrointestinal tract of various species, particularly within the lower esophageal sphincter. The pharmacological actions of the peptide suggest a role in the control of nonvascular smooth muscle tone.