Transport of the organic osmolyte betaine was characterized in brush-border membrane vesicles (BBMV) isolated from rabbit renal cortex. Inwardly directed gradients of either Na+ or H+ supported concentrative uptake in a manner consistent with the presence of parallel Na(+)-betaine and H(+)-betaine cotransport processes. Concentrative uptake occurred in the presence of membrane potential alone, indicating that betaine transport is electrogenic. Accumulation of betaine was not dependent on chloride in the medium. Whereas L-proline inhibited both the H(+)- and Na(+)-sensitive components of betaine transport, glycine blocked the H(+)-sensitive pathway and had little effect on Na(+)-sensitive betaine transport. Both pathways were adequately described by Michaelis-Menten kinetics. Under Na(+)-gradient conditions (pH equilibrium), the maximal rate of total betaine transport (Jmax) = 50.8 +/- 13.3 nmol.mg-1.min-1 and the concentration of total betaine producing half-maximal uptake (Kt) = 4.1 +/- 0.5 mM. Under H(+)-gradient conditions (Na+ free), Jmax = 102.5 +/- 10.5 nmol.mg-1.min-1 and Kt = 2.8 +/- 0.3 mM. Imposition of both Na+ and H+ gradients increased Jmax (142 +/- 25.5 nmol.mg-1.min-1) to a level significantly greater than that noted in the presence of a Na+ gradient alone. We conclude that betaine transport in renal BBMV involves two distinct transport pathways that are differentiated on the basis of sensitivity to either Na+ or H+ and by their specificity to proline and glycine.