In rabbit gallbladder epithelium, a Na+/H+, Cl-/HCO3- double exchange and a Na(+)-Cl- symport are both present, but experiments on intact tissue cannot resolve whether the two transport systems operate simultaneously. Thus, isolated apical plasma membrane vesicles were prepared. After preloading with Na+, injection into a sodium-free medium caused a stable intravesicular acidification (monitored with the acridine orange fluorescence quenching method) that was reversed by Na+ addition to the external solution. Although to a lesser extent, acidification took place also in experiments with an electric potential difference (PD) equal to 0. If a preset pH difference (delta pH) was imposed [( H+]in greater than [H+]out, PD = 0), the addition of Na-gluconate to the external solution caused delta pH dissipation at a rate that followed saturation kinetics. Amiloride (10(-4) M) reduced the delta pH dissipation rate. Taken together, these data indicate the presence of Na+ and H+ conductances in addition to an amiloride-sensitive, electroneutral Na+/H+ exchange. An inwardly directed [Cl-] gradient (PD = 0) did not induce intravesicular acidification. Therefore, in this preparation, there was no evidence for the presence of a Cl-/OH- exchange. When both [Na+] and [Cl-] gradients (outwardly directed, PD = 0) were present, fluorescence quenching reached a maximum 20-30 sec after vesicle injection and then quickly decreased. The decrease was not observed in the presence of a [Na+] gradient alone or the same [Na+] gradient with Cl- at equal concentrations at both sides. Similarly, the decrease was abolished in the presence of both Na+ and Cl- concentration gradients and hydrochlorothiazide (5 x 10(-4) M). The decrease was not influenced by an inhibitor of Cl-/OH- exchange (10(-4) M furosemide) or of Na(+)-K(+)-2Cl- symport (10(-5) M bumetanide). We conclude that a Na+/H+ exchange and a Na(+)-Cl- symport are present and act simultaneously. This suggests that in intact tissue the Na(+)-Cl- symport is also likely to work in parallel with the Na+/H+ exchange and does not represent an induced homeostatic reaction of the epithelium when Na+/H+ exchange is inhibited.