Background and Aims: Functional classes of enteric neurons in small mammals can be determined by neurochemical coding and axonal projections. The aim of this study was to describe the projections of vasoactive intestinal peptide (VIP)-, tachykinin (TK)-, and calretinin-immunoreactive (IR) interneurons and circular muscle motor neurons in human colon. Methods: Human colonic myenteric neurons were retrogradely filled with 1,1'-didodecyl 3,3,3',3'-indocarbocyanine perchlorate (Dil) from either the myenteric plexus or the circular muscle layers in organotypic culture preparations. The preparations were then labeled with antisera, and the position and immunoreactivity of Dil-filled neurons was recorded. Results: Nine percent of circular muscle motor neurons were labeled with TK immunoreactivity and these projected orally; 22% were VIP-IR and projected anally. Fifty-one percent of myenteric neurons that projected anally to the myenteric ganglia were VIP-IR and none were TK-IR. In contrast, 23% of neurons projecting orally were TK- IR but only 2% were VIP-IR. Calretinin immunoreactivity was present in 23% of neurons projecting anally and 3% projecting orally within the myenteric plexus. Six percent of circular muscle motor neurons had Dogiel type II morphology, and 50% of these were TK-IR. Conclusions: Circular muscle motor neurons and myenteric interneurons with TK, VIP, and calretinin immunoreactivity in the human colon have distinct projections and polarity.