The neuropeptide substance P (SP) is involved in the regulation of epithelial secretion and motility in the rat small intestine. The morphology, chemical profiles and proportion of SP-containing enteric neurons in this tissue have been examined by immunohistochemical analysis of whole-mount preparations obtained from colchicine-treated rats. In the submucosal plexus of the duodenum, jejunum and ileum, the proportion of SP-positive neurons is 53%, 51% and 49%, respectively. All SP-positive submucosal neurons are positive for neurofilament 200 (NF-200) and calretinin. Immunoreactivity for calcitonin gene-related peptide (CGRP) is detectable in 55% of the SP-positive submucosal neurons. Some SP-positive submucosal neurons have two or more long processes emerging from an oval or round cell body, a characteristic of the Dogiel type II neuron (type II neuron; a putative intrinsic primary afferent neuron). About one-third of the neurons in the myenteric plexus are positive for SP and a majority of them are NF-200/calretinin-positive type II neurons. Immunoreactivity for the SP receptor neurokinin-1 receptor (NK1R) has been detected mainly in the submucosal and myenteric NF-200-positive neurons, which are expected to contain SP. These neurons possibly stimulate each other via SP release. Most of the submucosal and myenteric neurons, including type II neurons, show immunoreactive for the prostaglandin E2 receptor EP3 receptor (EP3R). Thus, SP/NF-200/calretinin/NK1R/EP3R is the common chemical profile of type II neurons in the rat small intestine. The proportion of SP-immunopositive submucosal neurons (49%-53%) is higher in the rat small intestine than in the colon (≤11%) and around 50% are positive for CGRP.