Intracellular pH (pHi) of the acinar cells of the isolated, superfused mouse lacrimal gland has been measured using pH-sensitive microelectrodes. Under nonstimulated condition pHi was 7.25, which was about 0.5 unit higher than the equilibrium pH. Alterations of the external pH by +/- 0.4 unit shifted pHi only by +/- 0.08 unit. The intracellular buffering value determined by applications of 25 mM NH4+ and bicarbonate buffer solution gassed with 5% CO2/95% O2 was 26 and 46 mM/pH, respectively. Stimulation with 1 microM acetylcholine (ACh) caused a transient, small decrease and then a sustained increase in pHi. In the presence of amiloride (0.1 mM) or the absence of Na+, application of ACh caused a significant decrease in pHi and removal of amiloride or replacement with Na+-containing saline, respectively, rapidly increased the pHi. Pretreatment with DIDS (0.2 mM) did not change the pHi of the nonstimulated conditions; however, it significantly enhanced the increase in pHi induced by ACh. The present results showed that (i) there is an active acid extrusion mechanism that is stimulated by ACh; (ii) stimulation with ACh enhances the rate of acid production in the acinar cells; and (iii) the acid extrusion mechanism is inhibited by amiloride addition to and Na+ removal from the bath solution. We suggest that both Na+/H+ and HCO3-/Cl- exchange transport mechanisms are taking roles in the intracellular pH regulation in the lacrimal gland acinar cells.