Myenteric neurons projecting to the mucosa of the guinea pig proximal colon were identified using the combination of a neuronal tracing method and immunohistochemical techniques. The tracer DiI (1, 1'didodecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate) was applied onto the mucosa of a specimen of proximal colon which was then placed in organotypic culture to allow retrograde transport of the dye. After culture, the myenteric plexus was stained with antisera raised against choline acetyltransferase (ChAT) and calbindin (Calb). Of the myenteric neurons labeled with DiI, 99% had smooth cell bodies with Dogiel Type II morphology. Of these neurons, 70% projected in the longitudinal direction and the majority of them (65%) were located anally from the DiI application site, i.e., had ascending projections. Ascending neurons projected over significantly shorter distances than descending ones (3.1+/-0.5 mm vs. 4.6+/-1.2 mm, respectively; P<0.01). Of the labeled myenteric neurons, 98% were ChAT immunoreactive. Of these neurons, 78% were also immunoreactive for Calb and were preferentially ascending neurons. ChAT-immunoreactive but Calb-negative neurons did not have preferential projection. This study revealed the presence of two populations of myenteric neurons projecting to the mucosa of the guinea pig proximal colon. Morphological characteristics and neurochemical coding were suggestive for a putative sensory function for these neurons.