The current studies examine the presence of the Na⁺-HCO₃ ⁻ cotransporter in chicken enterocytes and its role in cytosolic pH (pH_i) regulation. The pH-sensitive dye 2′,7′-bis(carboxyethyl)-5,6-carboxy-fluorescein (BCECF) was used to monitor pH_i. Under resting conditions, pH_i was 7.25 in solutions buffered with bis(2-hydroxyethyl)-1-piperazine ethanesulphonic acid (HEPES) and 7.17 in those buffered with HCO₃ ⁻. Removal of external Na⁺ decreased pH_i and readdition of Na⁺ rapidly increased pH_i towards the control values. These Na⁺-dependent changes were greater in HCO ₃ ⁻ than in HEPES-buffered solutions. In HCO ₃ ⁻ - free solutions the Na⁺-dependent changes in pH_i were prevented by 5-(N-ethyl-N-isopropyl)-amiloride (EIPA) and unaffected by 4,4′-diisothiocyanatostilbene disulphonic acid (H₂-DIDS). In the presence of HCO ₃ ⁻ , the Na⁺-induced changes in pH_i were sensitive to both EIPA and H₂-DIDS. In the presence of EIPA, cells partially recovered from a moderate acid load only when both Na⁺ and HCO ₃ ⁻ were present. This pH_i recovery, which was EIPA resistant, and dependent on Na⁺ and HCO ₃ ⁻ , was inhibited by H₂-DIDS and occurred at equal rates in both Cl⁻-containing and Cl⁻-free solutions. Kinetic analysis of the rate of HCO ₃ ⁻ - and Na⁺- dependent pH_i recovery from an acid load as a function of the Na⁺ concentration revealed first-order kinetics with a Michaelis constant, K _m, of 11 mmol/l Na⁺. It is concluded that in HCO₃ ^/− buffered solutions both the Na⁺/H⁺ exchanger and the Na⁺-HCO₃ ⁻ cotransporter participate in setting the resting pH_i in isolated chicken enterocytes and help the recovery from acid loads.