The Flinders sensitive line (FSL) rats exhibit an increased cholinergic responsiveness in vivo when compared to their counterparts, the Flinders resistant line (FRL) rats. The functional consequences of this phenotypic difference on colonic mucosal function are not known. We sought to determine whether isolated distal colonic mucosa from the two strains exhibit differential responses to cholinergic agonists. The responses of the distal colonic mucosa from two lines of rats to carbachol were compared by recording changes in short‐circuit current. The ion movements associated with these changes were assessed by flux analysis of the radiotracers, 22Na and 36Cl. The anticipated hyper‐responsiveness to cholinergic stimulation in FSL rats was not seen. Carbachol responses were significantly enhanced by indomethacin pretreatment only in FRL rats. Tetrodotoxin (TTX) pretreatment significantly reduced responses to carbachol in FSL rats at all concentrations tested, though this was only seen with lower concentrations in FRL rats. Flux analysis indicated that both lines absorbed Na+ and Cl− under basal conditions and that a significant residual flux was present. Stimulation with carbachol led to significant reductions in net Na+ and Cl− fluxes in both lines. The changes in net Na+ and Cl− flux in both lines stem largely from a decrease in mucosal to serosal fluxes of both ions with an increase in serosal to mucosal flux of Cl−. The striking difference is the significant reduction in residual flux seen only in FRL rats. Indomethacin pretreatment abolished the changes in residual flux seen in FRL rats. Thus the responses to carbachol in these rats had at least three components: (a) a direct effect on the transporting colonocyte, (b) an indirect effect mediated by an arachidonic acid metabolite, and (c) another indirect effect involving a neurotransmitter. The relative contributions of each of these components were different in the two lines.