Cocaine and amphetamine regulated transcript (CART) encodes a novel brain-enriched protein whose features are reminiscent of a neurotransmitter propeptide. We have now localized CART peptide(s) in the gastrointestinal tract by immunohistochemical methods. Polyclonal antisera raised to CART peptide 106-129 stained neuronal cell bodies and fibers in rat ileum myenteric plexus-longitudinal muscle tissue preparations. Electron microscopic analysis of thin sections showed immunopositive axon terminals in close apposition to CART-labelled and unlabelled neuronal cell bodies as well as to the longitudinal muscle. CART peptide-immunoreactive terminals contained numerous ovoid electron-lucent vesicles and a few dark-stained dense-core vesicles. Light microscopic double labelling studies revealed CART peptide immunoreactivity in a subpopulation of choline acetyltransferase (ChAT)-immunoreactive neurons. This combined light microscopic and ultrastructural examination of CART peptides in the gastrointestinal tract suggests a role of CART peptides as transmitters or neuromodulators in the peripheral nervous system.