The sodium bicarbonate cotransporter (NBC) protein is functionally expressed in salivary glands. In this experiment, we examined the role of NBC in HCO₃-formation in human parotid gland acinar cells. Intracellular pH (pHi) was measured in 2'-7'-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF)-loaded cells. Acetazolamide (0.1 mM) and 4,4'-diisothio cyanatostilbene-2,2'-disulphonic acid (DIDS, 0.5 mM) were used as specific inhibitors of carbonic anhydrase and NBC, respectively. The degree of inhibition was assessed by measuring the pHi recovery rate (△pHi/min) after cell acidification using an ammonium prepulse technique. In control experiments, △pHi/min was 1.40±0.06. Treatment of cells with 0.5 mM DIDS or 0.1 mM acetazolamide significantly reduced △pHi/min to 1.14±0.14 and 0.74±0.15, respectively. Simultaneous application of DIDS and acetazolamide further reduced △pHi/min to 0.47±0.10. Therefore, DIDS and acetazolamide reduced △pHi/min by 19% and 47%, respectively, while simultaneous application of both DIDS and acetazolamide caused a reduction in △pHi/min of 67%. These results suggest that in addition to carbonic anhydrase, NBC also partially contributes to HCO₃- formation in human parotid gland acinar cells.