Functional expression of the rat colonic H+-K+-ATPase was obtained by coexpressing its catalytic α-subunit and the β1-subunit of the Na+-K+-ATPase in Xenopus laevis oocytes. We observed that, in oocytes expressing the rat colonic H+-K+-ATPase but not in control oocytes (expressing β1 alone), NH4Cl induced a decrease in86Rb uptake and the initial rate of intracellular acidification induced by extracellular NH4Cl was enhanced, consistent with [Formula: see text] influx via the colonic H+-K+-ATPase. In the absence of extracellular K+, only oocytes expressing the colonic H+-K+-ATPase were able to acidify an extracellular medium supplemented with NH4Cl. In the absence of extracellular K+ and in the presence of extracellular [Formula: see text], intracellular Na+ activity in oocytes expressing the colonic H+-K+-ATPase was lower than that in control oocytes. A kinetic analysis of86Rb uptake suggests that[Formula: see text] acts as a competitive inhibitor of the pump. Taken together, these results are consistent with[Formula: see text] competition for K+ on the external site of the colonic H+-K+-ATPase and with [Formula: see text] transport mediated by this pump.