Pancreatic beta-cells are known to maintain intracellular pH (pHi) at a value well above that predicted from the electrochemical gradient. The mechanisms for the active extrusion of protons were examined by continuously monitoring pHi in individual beta-cells from ob/ob mice using the fluorescent indicator 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF). In a medium nominally devoid of bicarbonate, the steady-state pHi was 6.82 +/- 0.02 and the intracellular buffering capacity was equivalent to 79 +/- 3 mM/pH unit. pHi remained unaffected after raising the glucose concentration from 3 to 20 mM, it was lowered when depolarizing the beta-cells with tolbutamide and it increased in the presence of carbachol. After removal of Na+ there was a significant drop of pHi and blockage of the pHi recovery following acid loading with the NH4+ prepulse technique. Whereas addition of amiloride had a similar, but less pronounced effect, omission of Cl- resulted in moderate alkalinisation. After switching to a medium containing bicarbonate, minor acidification was followed by adjustment of pHi to a steady state higher than the initial one. The results indicate that the acid load arising from glucose metabolism in the beta-cells is effectively buffered and the protons extruded both by Na+-H+ and Cl- -HCO3- exchangers.