The effect of carbachol, an agonist of the Ca2+ pathway, on K+ transport in rat proximal and distal colon was studied by measuring unidirectional fluxes, uptake, and efflux of Rb+, a marker for K+, in mucosa-submucosa preparations. Unidirectional ion flux measurements revealed that carbachol stimulated K+ secretion in the proximal colon by a marked increase in the serosa-to-mucosa flux (J(Rb)sm) and a more moderate rise in the mucosa-to-serosa flux (J(Rb)ms). In the distal colon carbachol had no effect on J(Rb)ms but J(Rb)sm was reduced after a transient increase finally resulting in an inhibition of K+ secretion. Carbachol caused a stimulation of mucosal Rb+ uptake in the distal colon, which was diminished in the presence of inhibitors of the apical H+-K+-ATPase, vanadate and ouabain. In contrast, in the proximal colon the serosal Rb+ uptake was enhanced by carbachol, an effect, which could be prevented by bumetanide, an inhibitor of the basolateral Na+-K+-2Cl(-)-cotransporter. Efflux experiments revealed that carbachol caused a transient increase of apical and basolateral Rb+ permeability in both colonic segments. In the distal colon, stimulated K+ efflux to the serosal side was reduced by quinine, efflux to the mucosal side was blocked by tetraethylammonium. In the proximal colon, carbachol-activated apical and basolateral K+ efflux were inhibited by Ba2+. In conclusion, these data suggest that in the distal colon carbachol stimulates the H+-K+-ATPase and the basolateral K+ efflux through quinine-sensitive K+ channels, whereas in the proximal colon carbachol induces K+ secretion due to a stimulation of the basolateral Na+-K+-2Cl(-)-cotransporter and an increased efflux to the luminal side via Ba2+-sensitive apical K+ channels.