The aim of this study was to identify myenteric pathways to the circular and longitudinal muscle of the guinea pig proximal colon. To identify excitatory and inhibitory muscle motoneurones, we applied the neuronal retrograde tracer DiI onto the circular or longitudinal muscle layer and performed additional immunohistochemistry for nitric oxide synthase (NOS) and choline acetyltransferase (ChAT). On average 166 +/- 81 circular muscle motoneurones (CMMN) and 100 +/- 74 longitudinal muscle motoneurones (LMMN) were labelled by DiI tracing. Myenteric pathways innervating the muscle were either ascending (DiI-labelled neurones with oral projections) or descending (DiI-labelled neurones with anal projections). The circular muscle was preferentially innervated by ascending pathways (66.0 +/- 9.1%). Most ascending CMMN were ChAT-positive (87.2 +/- 8.5%), whereas descending CMMN were mainly NOS-positive (82.3 +/- 14.6%). Most ascending (62.2 +/- 11.1%) and descending (82.0 +/- 12.5%) CMMN had circumferential projection preferences (circumferential projections were longer than projections along the longitudinal gut axis). In contrast to the polarised projections to the circular muscle, the longitudinal muscle was equally innervated by ascending (46.2 +/- 15.1%) and descending (53.9 +/- 15.1%) neurones. Ascending and descending pathways to the longitudinal muscle consisted predominantly of ChAT-positive neurones (98.1 +/- 1.9% and 68.0 +/- 8.5%, respectively), and both pathways had prominent longitudinal projection preferences. Only 25.5% of the descending LMMN were NOS-positive. In conclusion, the circular muscle in the proximal colon is innervated by descending inhibitory (NOS-positive neurones) and ascending excitatory (ChAT-positive neurones) pathways. In contrast, the longitudinal muscle is primarily innervated by ascending and descending excitatory motoneurones, and only a small proportion of the descending pathway consisted of inhibitory motoneurones.