Transport interactions among phosphate, glucose, and alanine were evaluated in brush border membrane vesicles isolated from the rabbit renal cortex. Both glucose and alanine inhibited the Na+ gradient-stimulated uphill accumulation of phosphate. Neither glucose nor alanine inhibited the phosphate uptake measured in the absence of Na+ or in the presence of a collapsed Na+ gradient shunted with amphotericin B. Phosphate and alanine inhibited the Na+ gradient-stimulated uphill accumulation of glucose but not the glucose uptake measured in the presence of a collapsed Na+ gradient shunted with amphotericin B. Alanine, which was more potent than glucose or phosphate in inhibiting Na+ gradient-stimulated solute transport, was also more potent than glucose or phosphate in stimulating the uptake of Na+ into the vesicles. We conclude that the inhibitory interactions among phosphate, glucose, and alanine probably represent indirect effects resulting from solute-induced alterations in the transmembrane electrochemical Na+ gradient rather than direct effects resulting from competition for a polyfunctional carrier or from allosteric interactions. Such a mechanism could explain the inhibitory interactions among phosphate, glucose, and alanine that have been observed in the intact proximal tubule.