The CO(2)/HCO(3)(-) buffering system is one of the main mechanisms implicated in cytosolic pH (pH(i)) regulation. We studied this pH(i)-regulatory system in rat mast cells using a fluorescent dye. Mast cells had a more alkaline pH(i) in the presence of HCO(3)(-) than in its absence. The recovery from an acid load was faster in HCO(3)(-)-free conditions than in HCO(3)(-)-containing media. In HCO(3)(-)-buffered conditions the increase of the recovery rate of an acidification in 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid-incubated cells suggested the implication of a Na(+)-independent Cl(-)/HCO(3)(-) exchanger. This HCO(3)(-) transport acidified the cytosol and was also partially responsible for the recovery of intracellular alkalinizations. Moreover, regulation of the recovery rate of an acidification by protein kinase C and calcium signaling pathways depended on the presence or absence of HCO(3)(-). The presence of HCO(3)(-) limits the recovery of acute intracellular acidifications probably through the Na(+)-independent Cl(-)/HCO(3)(-) exchanger and modulates the regulation of pH(i) by protein kinase C and calcium.