This study establishes the characteristics of electroneutral Na-H exchange present in apical membrane vesicles isolated from colonocytes of rat large intestine. An outward directed proton gradient stimulated 22Na uptake and demonstrated transient accumulation that was not altered by voltage clamping with K and its ionophore, valinomycin. Amiloride inhibited proton gradient-stimulated 22Na (0.1 mM) uptake by 97% by a noncompetitive mechanism with a Ki of 27 microM. 5-(N-ethyl-N-isopropyl)amiloride, an amiloride analogue which is a specific potent inhibitor of electroneutral Na-H exchange, inhibited proton gradient-stimulated 22Na uptake with a Ki of 400 nM. Increasing Na concentration saturated the proton gradient-stimulated 22Na uptake with an apparent Km of 11.7 +/- 2.2 mM. Increasing intravesicular proton concentration also resulted in saturation of 22Na uptake with a Km of 2.8 microM. Proton gradient-stimulated 22Na uptake was inhibited significantly by 10 mM Na+, Li+, and NH4+, while outward directed gradients of these ions in the absence of proton gradient accelerated 22Na uptake. The mechanism of Li+ inhibition of proton gradient-stimulated 22Na uptake was competitive with a Ki of 2 mM. 22Na uptake either stimulated by an outward directed Na gradient (i.e. Na-Na exchange) at pH 5.5, 6.5, and 7.5 or stimulated by 10-fold outward directed proton gradient at different pH values was similar; observations that do not suggest proton activation of Na-H exchange. We conclude from these results that proton gradient-stimulated 22Na uptake in colonic apical membrane vesicles represents tightly coupled electro-neutral Na-H exchange with several kinetic properties unlike those described in other epithelia.