The involvement of Na(+)-H+ exchange in chicken colonocyte homeostasis was investigated. Colonocyte pH (pH(i)) was measured with 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein (BCECF). The proton ionophore FCCP reduced basal pH(i), indicating that cytosolic [H+] is not at electrochemical equilibrium across the membrane. External Na+ removal decreased pH(i) and subsequent addition of Na+ returns pH(i) towards its control value. The rate of pH(i) recovery from an acid load was Na(+)-dependent (K(m) for Na+, 24 mM) and inhibited by EIPA (IC50, 0.18 microM). The initial rate of Na(+)-dependent cell alkalization increased as the pH(i) decreased from 7.2 to 6.6 (Hill coefficient, 1.88). Radioisotope flux studies revealed that an outwardly directed proton gradient transiently stimulated Na+ uptake into BBMV isolated from the chick colon. EIPA and amiloride inhibited pH gradient-driven Na+ uptake (IC50 of 4 microM and 32 microM, respectively). The K(m) for Na+ of pH gradient-driven Na+ uptake was 6.8 mM. The Hill coefficient of the relationship between the initial rate of pH-driven Na+ uptake and the intravesicular pH was 0.70. It is concluded that a Na(+)-H+ exchanger is involved in pH(i) homeostasis in chicken colonocytes and that these cells possess at least two types of Na(+)-H+ antiporters with different sensitivity to EIPA and different kinetic parameters.