1. Intracellular pH (pHi) plays an important role in regulating fluid and electrolyte secretion by salivary gland acinar cells. The pH-sensitive, fluorescent dye 2', 7'-bis(carboxyethyl)-5(6)-carboxylfluorescein (BCECF) was used to characterize the mechanisms involved in regulating pHi during muscarinic stimulation in mouse sublingual mucous acinar cells. 2. In the presence of HCO3-, muscarinic stimulation caused a rapid decrease in pHi (0.24 +/- 0.02 pH units) followed by a slow recovery rate (0.042 +/- 0.002 pH units min-1) to the initial resting pHi in sublingual acinar cells. The muscarinic receptor-induced acidification in parotid acinar cells was of a similar magnitude (0. 25 +/- 0.02 pH units), but in contrast, the recovery rate was approximately 4-fold faster (0.181 +/- 0.005 pH units min-1). 3. The agonist-induced intracellular acidification was inhibited by the anion channel blocker niflumate, and was prevented in the absence of HCO3- by treatment with the carbonic anhydrase inhibitor methazolamide. These results indicate that the muscarinic-induced acidification is due to HCO3- loss, probably mediated by an anion conductive pathway. 4. The Na+-H+ exchange inhibitor 5-(N-ethyl-N-isopropyl)amiloride (EIPA) amplified the magnitude of the agonist-induced acidification and completely blocked the Na+-dependent pHi recovery. 5. To examine the molecular nature of the Na+-H+ exchange mechanism in sublingual acinar cells, pH regulation was investigated in mice lacking Na+-H+ exchanger isoforms 1 and 2 (NHE1 and NHE2, respectively). The magnitude and the rate of pHi recovery in response to an acid load in acinar cells isolated from mice lacking NHE2 were comparable to that observed in cells from wild-type animals. In contrast, targeted disruption of the Nhe1 gene completely abolished pHi recovery from an acid load. These results demonstrate that NHE1 is critical for regulating pHi during a muscarinic agonist-stimulated acid challenge and probably plays an important role in regulating fluid secretion in the sublingual exocrine gland. 6. In NHE1-deficient mice, sublingual acinar cells failed to recover from an acid load in the presence of bicarbonate. These results confirm that the major regulatory mechanism involved in pHi recovery from an acid load is not Na+-HCO3- cotransport, but amiloride-sensitive Na+-H+ exchange via isoform 1.