The findings (a) stereospecific counter transport; (b) equilibrium uptake of L-proline decreased with increasing medium osmolarity; and (c) L-proline and Na+ were taken up into identical intravesicular spaces, indicate that uptake of L-proline by rabbit renal brush border membranes represents transport into membrane vesicles, not surface binding to the membrane. An Na+ gradient between extravesicular and intravesicular media stimulated the initial rate of L-proline uptake about 10 times. Accumulation of the imino acid was maximal at 2 min, then decreased until the equilibrium level was attained. At the peak of this "overshoot" uptake of L-proline was 3-fold greater than the final equilibrium value. These results suggest that the electrochemical Na+ gradient drives the transient movement of L-proline into the membrane vesicles against its concentration gradient. Stimulation of L-proline uptake was specific for Na+. Increasing the Na+ gradient lowered the apparent Km for L-proline. Efflux of L-proline from the membrane vesicles, like uptake, showed stimulatory effects when the Na+ gradient and L-proline were on the same side, and inhibitory effects when the Na+ gradient and the imino acid were on opposite sides of the membrane. Uptake of L-proline, at a given concentration, reflected the sum of contributions from Na+ gradient-dependent and -independent transport systems. The dependent system was saturated at 4 mM L-proline. The independent system did not saturate but may represent the sum of passive diffusion and a "carrier"-mediated system. At physiological concentrations the rate of the Na+ gradient-dependent uptake was 5 times that in the absence of the gradient. In K+-loaded vesicles, valinomycin, but not nigericin, enhanced the Na+ gradient-dependent uptake of L-proline. Gramicidin diminished uptake. These findings indicate that the Na+ gradient-dependent transport of L-proline is an electrogenic process and suggest that the membrane potential is a determinant of L-proline transport. The Na+ gradient-dependent rate of L-proline uptake was strongly inhibited by other imino acids, suggesting that L-imino acids have a common transport system. Glycine and neutral amino acids inhibited the rate of L-proline uptake moderately, acidic amino acid and D-glucose were very weak inhibitors, and basic amino acids were without effect. In the absence of Na+, the rate of L-proline uptake was independent of the presence of other amino acids. These findings indicate that the brush border membrane is a site of amino acid recognition during vectorial transepithelial transport. It is proposed that imino and neutral amino acids inhibit transport of L-proline by competitive interaction with the L-proline "carriers" in addition to competition for the electrochemical Na+ gradient or membrane potential. The findings are relevant to the understanding of genetic amino acid transport disorders, such as iminoglycinuria.