Immunofluorescence was used to study immunoreactivity (IR) for corticotropin-releasing factor (CRF) in the guinea pig enteric nervous system. CRF-IR was expressed in both the myenteric and the submucosal plexuses of all regions of the large and small intestine and the myenteric plexus of the stomach. CRF-IR nerve fibers were present in the myenteric and submucosal plexuses, in the circular muscle coat, and surrounding submucosal arterioles. Most of the CRF-IR fibers persisted in the myenteric and submucosal plexuses after 7 days in organotypic culture. CRF-IR was not coexpressed with tyrosine hydroxylase-IR or calcitonin gene-related peptide-IR fibers. The proportions of CRF-IR cell bodies in the myenteric plexus increased progressively from the stomach (0.6%) to the distal colon (2.8%). Most of the CRF-IR myenteric neurons (95%) had uniaxonal morphology; the remainder had Dogiel type II multipolar morphology. CRF-IR cell bodies in the myenteric plexus of the ileum expressed IR for choline acetyltransferase (56.9%), substance P (55.0%), and nitric oxide synthase (37.9%). CRF-IR never colocalized with IR for calbindin, calretinin, neuropeptide Y, serotonin, or somatostatin in the myenteric plexus. CRF-IR cell bodies were more abundant in the submucosal plexus (29.9-38.0%) than in the myenteric plexus. All CRF-IR neurons in submucosal ganglia expressed vasoactive intestinal peptide-IR and were likely to be secretomotor/vasodilator neurons. CRF-IR neurons did not express IR for the CRF(1) receptor. CRF(1)-IR was expressed in neuronal neighbors of those with CRF-IR. Collective evidence suggests that VIPergic secretomotor neurons might provide synaptic input to neighboring cholinergic neurons.