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 Na-22 uptake. Proton gradient-stimulated Na-22 uptake was activated severalfold by the additional presence of an inward bicarbonate gradient, and bicarbonate gradient-stimulated Na-22 uptake was significantly enhanced by an imposed intravesicular membrane positive potential. 0.1 mM amiloride inhibited both proton gradient-and bicarbonate gradient-stimulated Na-22 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 Na-22 uptake by 40 and 80%, respectively. Both proton gradient- and bicarbonate gradient-stimulated Na-22 uptake saturated as a function of increasing Na concentration: the apparent kinetic constants (K(m)) for Na for the DIDS-insensitive component of proton gradient-stimulated Na-22 uptake was 46.4 mM, while the DIDS-sensitive component of proton gradient- and bicarbonate gradient-stimulated Na-22 uptake had K(m) for Na of 8.1 and 6.4 mM, respectively. Amiloride inhibited both DIDS-insensitive proton gradient- and bicarbonate gradient-stimulated Na-22 uptake with an inhibitory constant (K(i)) of approximately 35 and 1-mu-M, respectively. We conclude from these results that proton graident-stimulated Na-22 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 Na-22 uptake and bicarbonate gradient-stimulated Na-22 uptake may represent the same electrogenic Na-anion cotransport process.