Stimulation of cells of the rat basophilic leukemia line RBL-2H3, which are used as a model in biochemical studies of mast cells, by antigen or by the calcium ionophore ionomycin, are known to cause secretion of mediators of inflammation. These stimuli have now been found to cause a decrease in the cells' cytosolic pH. This acidification process was monitored by the fluorescent indicator 2',7'-bis (carboxyethyl)-5(6)-carboxyfluorescein (BCECF) introduced into these cells. The antigen induced acidification was the result of specific aggregation of membrane residing IgE, reached values up to 0.03 pH units and required the presence of sodium and calcium ions in the incubation medium. It was amiloride resistant but was blocked by the metabolic inhibitor deoxyglucose. Ionomycin caused a dose dependent decrease in cytosolic pH which was also sensitive to the pH of the extracellular medium. The acidification reached more than 0.1 pH units at optimal, non-cytotoxic, doses of ionomycin (1 microM) and decreased markedly as the medium pH increased from 7.0 to 8.0. The antigen and ionophore induced cytosolic acidification processes are interpreted as being the result of the increased concns of free cytosolic calcium ions rather than the effect of direct activation of a sodium-proton exchanger. Further investigation of this process is in progress.