The major pathway for HCO3 transport across the basolateral membrane of the proximal tubule cell is electrogenic Na'-HCO-cotransport. In this study, we have determined the stoichiometry of the Na'-HCO-cotransport system in basolateral membrane vesicles that were isolated from rabbit renal cortex by Percoll gradient centrifugation. When the membrane potential is approximated by the Nernst potential for K+, as in the presence of the K+ ionophore valinomycin, equilibrium thermodynamics predicts that the Na'-HCO3 cotransport system should come to equilibrium and mediate no net flux when (Nas/ (Na)0 = I(HCO3)./(HCO3)d"I(K)o/(K)1F', where n is the HCO-:Na' stoichiometry. Our experimental approach was to impose transmembrane Na+, HCO3, and K+ gradients of varying magnitude and direction, and then to measure the net flux of Na+ over the subsequent 3-s period. In this way, we could determine the conditions for equilibrium of the transport system and thereby calculate n. The results of these experiments indicate that the value of n is > 2.6 and < 3.5, consistent with a stoichiometry of 3 HCO3 :1 Na+, or a thermodynamically equivalent process. Based on reported intracellular potentials and ion activities, this value for the stoichiometry indicates that the insidenegative membrane potential is sufficient to drive HCO3 exit against the inward concentration gradients of HCO3 and Na+ that are present across the basolateral membrane of the intact proximal tubule cell under physiologic conditions.