The nerve fibers that supply the external muscle of the human gastrointestinal tract were examined for their immunoreactivity to the neuropeptides enkephalin, neuropeptide Y, somatostatin, substance P, and vasoactive intestinal peptide, for tyrosine hydroxylase (a catecholamine-synthesizing enzyme), and for coexistence between immunoreactivities in nerve fibers. Studies on coexistence revealed that the majority of reactive nerve fibers could be placed in one of two classes: (a) those fibers with reactivity to enkephalin or substance P, or both, and (b) fibers containing one or both of the peptides neuropeptide Y and vasoactive intestinal peptide. Many fibers immunoreactive for vasoactive intestinal peptide or neuropeptide Y, or both, were found throughout the external smooth muscle of the gastrointestinal tract, but neuropeptide Y-reactive fibers were less common in the small and large intestines than in the stomach and esophagus. Fibers immunoreactive for enkephalin or substance P, or both, were sparse in the esophagus, increased in numbers to reach maximal frequency in the pylorus, and maintained a similar frequency in the small and large intestines. Fibers with somatostatin or tyrosine hydroxylase immunoreactivity were rare. In general, sphincter regions were similar to nonsphincter regions in peptide-immunoreactive fiber numbers and types, except that the internal anal sphincter had no enkephalin-immunoreactive fibers and very few substance P-reactive fibers. Moderate numbers of fibers reactive for neuropeptide Y and vasoactive intestinal peptide were found in the internal anal sphincter. It is suggested that enkephalin and substance P are in excitatory fibers and that vasoactive intestinal peptide and neuropeptide Y are in fibers inhibitory to the external muscle.