Background and Aims: Enteric neurons can be characterized by their chemical coding, projections, and morphology. The aim of this study was to describe the different classes of human colonic circular muscle motor neurons. Methods: Human colonic circular muscle motor neurons were identified by retrograde tracing with 1,1'-didodecyl 3,3,3',3'-indocarbocyanine perchlorate (Dil) applied to the circular muscle layer. Whole-mount preparations of the myenteric plexus were then double-labeled with antisera to choline acetyltransferase (CHAT) and/or nitric oxide synthase (NOS), or NOS and vasoactive intestinal peptide (VIP), and the position and immunoreactivity of Dil-filled neurons were recorded. Results: Fifty-two percent of all Dil-filled neurons were ChAT immunoreactive, and 86% of these projected up to 11 mm orally, with 14% projecting short distances anally. Forty-eight percent of the Dil-filled neurons were NOS immunoreactive, and 77% of these projected up to 19 mm anally, with 23% projecting no more than 6 mm orally. A subpopulation of these NOS-immunoreactive motor neurons were also VIP-immunoreactive. A small population of myenteric neurons was immunoreactive for both ChAT and NOS, but none projected to the circular muscle. NOS-immunoreactive motor neurons projected for longer distances than those with ChAT immunoreactivity and were larger. Conclusions: There are two classes of human colonic motor neurons: one is excitatory (ChAT- immunoreactive) and mainly projects orally and the other is inhibitory (NOS ± VIP immunoreactive) and projects preferentially anally.