The classification, morphology and function of enteric neurons have been extensively studied in the small and large intestine of the guinea pig. However, little is known about enteric neurons that directly project to the central nervous system (CNS). Previous studies have identified these unique neurons in the rectum, rectospinal neurons, but little was done to characterize them. Therefore, the aim of this study is to identify and characterize enteric neurons in the rat colon that directly project to the CNS by using retrograde neuronal tracing and immnohistochemistry. By applying the retrograde tracer DiI to the L6/S1 segments of the spinal cord, we identified these neurons in both the myenteric and submucosal plexus of the colon. These neurons expressed peptides associated with sensory neurons such as substance P (SP) and vasoactive intestinal polypeptide (VIP) but did not express calcitonin gene-related peptide (CGRP). The N-methyl-D-aspartate (NMDA) receptor subunits NR1 and NR2D and proteinase- activated receptor-2 (PAR2) were also found in these neurons. However they did not express the vanilloid receptor subtype 1(VR1) or neurokinin 1 receptor (NK1). The expression of the peptides and receptors suggests that there are at least two separate populations of neurons projecting from the colon to the CNS. The data indicate that these colospinal neurons are involved in pain transmission. The identification of colospinal afferent neurons (CANs) suggests that there is a direct link from the enteric to the central nervous system.