This study was undertaken to investigate the presence of a Na(+)-H(+) antiporter in brush-border membrane vesicles (BBMV) isolated from chick small intestine. An outwardly directed proton gradient (pH 5.5 inside, 7.5 outside) stimulated Na+ uptake into BBMV and resulted in a transient accumulation. No accumulation was observed in the absence of a proton gradient. Voltage clamping the membrane with K+ and valinomycin decreased the Na+ overshoot. Amiloride inhibited pH gradient-driven Na+ uptake in a dose-dependent manner with an IC50 of 44 microM. The relationship between pH gradient-driven Na+ uptake and external Na+ concentration followed simple, saturating Michaelis-Menten kinetics. Eadie-Hofstee analysis of the pH gradient-driven Na+ uptake indicated a single transport system with a Vmax of 33 nmol/mg protein per 15 s and a Km for Na+ of 12 mM. The initial rate of pH-driven Na+ uptake increased as the intravesicular pH decreased, with a Hill coefficient close to 1. These findings indicate that BBMV isolated from chicken small intestine possess a Na(+)-H(+) exchanger. This exchanger does not appear to be the one involved in cell pH regulation.