The major pathway for HCO3- transport across the basolateral membrane of the proximal tubule cell is electrogenic Na+-HCO3- cotransport. In this study, we have determined the stoichiometry of the Na+-HCO3- 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 (Na)i/(Na)o = [(HCO3)o/(HCO3)i]n[(K)o/(K)i]n-1, where n is the HCO3-: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 greater than 2.6 and less than 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 inside-negative 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.