The immunohistochemical localization of peptide F and the related enkephalins met5-enkephalin (met-enk), leu5-enkephalin (leu-enk), met5-enkephalin-arg6-phe7 (met-enk-arg-phe), and met5-enkephalin-arg6-gly7-leu8 (met-enk-arg-gly-leu) was investigated by means of the indirect immunofluorescence technique in the enteric nervous system of the rat. Peptide F-like immunoreactivity was widely distributed within neuronal structures throughout the gastrointestinal (GI) tract of the rat. Peptide F-containing nerve cell bodies were mainly located in the myenteric plexus, and only rarely were seen in the submucosal plexus. Peptide F-immunoreactive nerve fibers were principally present in the myenteric plexus and circular muscle layer; few were found in the submucosal plexus, longitudinal muscle layer, muscularis mucosa, and mucosa. No peptide F-containing fibers were found associated with blood vessels in the gut. By comparing the distribution of immunoreactive peptide F with other related enkephalins met-enk, leu-enk, met-enk-arg-phe, and met-enk-arg-gly-leu, we observed that there was a remarkable similarity in the distribution of peptide F and other enkephalins in the GI tract. These data, combined with our previous studies, indicate that peptide F may coexist with other related enkephalins in the same neurons of the enteric nervous system. The results suggest that peptide F, a product of the proenkephalin A gene, may play a physiological role within the enteric nervous system.