The transport of L-alanine was studied using membrane vesicles derived from the brush borders of the rabbit renal proximal tubule. Preincubation of the renal membranes with L-alanine, but not D-alanine, accelerated exchange diffusion of L-alanine, i.e. stereospecific counter transport. The equilibrium uptake of L-alanine decreased with increasing medium osmolarity. Extrapolation to infinite medium osmolarity, i.e. zero intravesicular space, indicates no uptake. These findings demonstrate that the uptake of L-alanine represents transport into membrane vesicles and not surface binding to the membrane. The presence of a Na+ gradient between the external incubation medium and the intravesicular medium stimulated L-alanine uptake. Accumulation of the amino acid in the vesicles was maximal at 5 min and then decreased, indicating efflux. The final level of uptake in the presence of the Na+ gradient was identical with that obtained in the absence of the gradient, suggesting that equilibrium was established. At the peak of the "overshoot" the uptake of L-alanine was slmost twice the final equilibrium value. These results suggest that the imposition of a large extravesicular to intravesicular gradient of Na+ effects the transient movement of L-alanine into renal brush border membrane vesicles against its concentration gradient. Stimulation of L-alanine uptake was specific for Na+. When the intravesicular medium contained no Na+, the rate of uptake enhanced with increases in the concentration of Na+ in the external medium. Increasing the Na+ gradient lowered the apparent Km for L-alanine. In the absence of the Na+ gradient, the rates of uptake of L- and D-alanine were identical. In the presence of the Na+ gradient, the rate of D-alanine uptake was stimulated, but significantly less than that of L-alanine. The uptake of L-alanine, at a given concentration of amino acid reflected the sum of the contributions from Na+ gradient-dependent and -independent transport systems. The dependent system was saturated at about 2 mM L-alanine. The independent system exhibited minimal saturability and may itself represent the sum of passive diffusion and a "carrier"-mediated system. At physiological concentrations of L-alanine, the rate of the Na+ gradient-dependent uptake was 5-fold that in the absence of the gradient. Valinomycin enhanced the Na+ gradient-dependent uptake of L-alanine, provided a K+ gradient (vesicle greater than medium) was present. This finding indicates that the Na+ gradient-dependent transport of L-alanine into renal brush border membrane vesicles is an electrogenic process and suggests that the membrane potential is a determinant of L-alanine transport. In the presence of a Na+ gradient, the uptake of L-alanine was strongly inhibited by other neutral L-amino-acids. Imino acids and glycine also inhibited, but acidic and basic amino acids were without effect. In the absence of a Na+ gradient, little selective competition was found...