We wished to establish the functional identity of Na(v)1.6-expressing myenteric neurons of the guinea pig proximal colon by determining the extent of colocalization of Na(v)1.6 and selected neurochemical markers. Na(v)1.6-like immunoreactivity (-li) was primarily localized to the hillock and initial segments of myenteric neurons located near junctions with internodal fiber tracts. Immunoreactivity for Na(v)1.6 was co-localized with choline-acetyltransferase-li, representing 96% of Na(v)1.6-immunoreactive neurons; about 5% of these neurons showed co-localization with calretinin-li, but none with substance-P-li. Cholinergic neurons expressing Na(v)1.6 were amongst the smallest (somal area <300 mum(2)) of all cholinergic myenteric neurons observed. Only three of 234 Na(v)1.6-immunoreactive neurons exhibited nNOS-li, and none co-localized with calbindin-li. These data suggest that Na(v)1.6 is expressed in a small uniform population of cholinergic myenteric neurons that lie within the guinea pig proximal colon and that are likely to function as excitatory motor neurons.