This study sought to characterize the mechanism of Na transport across basolateral membrane vesicles of rat distal colon. Both an outward proton gradient and an inward bicarbonate gradient stimulated 22Na uptake. Proton gradient-stimulated 22Na uptake was activated severalfold by the additional presence of an inward bicarbonate gradient, and bicarbonate gradient-stimulated 22Na uptake was significantly enhanced by an imposed intravesicular membrane positive potential. 0.1 mM amiloride inhibited both proton gradient- and bicarbonate gradient-stimulated 22Na uptake by 80 and 95%, respectively, while 1 mM 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) inhibited both proton gradient- and bicarbonate gradient-stimulated 22Na uptake by 40 and 80%, respectively. Both proton gradient- and bicarbonate gradient-stimulated 22Na uptake saturated as a function of increasing Na concentration: the apparent kinetic constants (Km) for Na for the DIDS-insensitive component of proton gradient-stimulated 22Na uptake was 46.4 mM, while the DIDS-sensitive component of proton gradient- and bicarbonate gradient-stimulated 22Na uptake had Km for Na of 8.1 and 6.4 mM, respectively. Amiloride inhibited both DIDS-insensitive proton gradient- and bicarbonate gradient-stimulated 22Na uptake with an inhibitory constant (Ki) of approximately 35 and 1 microM, respectively. We conclude from these results that proton gradient-stimulated 22Na uptake represents both DIDS-insensitive Na-H exchange and DIDS-sensitive electrogenic Na-OH cotransport, and that the DIDS-sensitive component of proton gradient-stimulated 22Na uptake and bicarbonate gradient-stimulated 22Na uptake may represent the same electrogenic Na-anion cotransport process.