Ionophore use an illustrated by a description of experiments in which the K+ ionophore valinomycin and the uncoupler, carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) were employed to investigate Na+ transport pathways in renal microvillus membrane vesicles. First, the potential-dependence of solute transport was examined by using valinomycin and K+ gradients to alter the membrane potential. Whereas Na+-glucose cotransport was voltage-sensitive, the transport of Na+ in the absence of glucose was found to occur via a voltage-insensitive process, likely Na+-H+ exchange. Second, FCCP short-circuiting of the membrane vesicles was used to examine possible electrical interactions among Na+ transport pathways. The alanine inhibition of Na+-dependent glucose transport was abolished by FCCP, indicating that the effect of the amino acid on sugar flux was indirect and mediated by an alteration in the membrane potential. Third, aspects of the molecular mechanism of Na+-glucose cotransport were evaluated by using ionophores to study the potential-dependence of phlorizin binding to the Na+-coupled sugar carrier. Whereas the rate of phlorizin association was potential-dependent, the rate of release of bound phlorizin was insensitive to variation in the transmembrane voltage, suggesting that the potential-dependence of Na+-glucose cotransport arises from potential-dependent behavior of the free carrier rather than from potential-dependence of Na+-glucose translocation per se. These studies demonstrate that the use of ionophores augments the value of employing isolated plasma membrane vesicles to investigate mechanisms of epithelial solute transport.