The proximal tubule Na+-HCO3- cotransporter is located in the basolateral plasma membrane and moves Na+, HCO3-, and net negative charge together out of the cell. The presence of charge transport implies that at least two HCO-3 anions are transported for each Na+ cation. The actual ratio is of physiological interest because it determines direction of net transport at a given membrane potential. To determine this ratio, a thermodynamic approach was employed that depends on measuring charge flux through the cotransporter under defined ion and electrical gradients across the basolateral plasma membrane. Cells from an immortalized rat proximal tubule line were grown as confluent monolayer on porous substrate and their luminal plasma membrane was permeabilized with amphotericin B. The electrical properties of these monolayers were measured in a Ussing chamber, and ion flux through the cotransporter was achieved by applying Na+ or HCO3- concentration gradients across the basolateral plasma membrane. Charge flux through the cotransporter was identified as difference current due to the reversible inhibitor dinitro-stilbene disulfonate. The cotransporter activity was Cl- independent; its conductance ranged between 0.12 and 0.23 mS/cm2 and was voltage independent between -60 and +40 mV. Reversal potentials obtained from current-voltage relations in the presence of Na+ gradients were fitted to the thermodynamic equivalent of the Nernst equation for coupled ion transport. The fit yielded a cotransport ratio of 3HCO3-:1Na+.