Calcium ionophores such as ionomycin and A23187 are often used to determine the role of intracellular Ca+ + in cellular processes. Ionomycin but not Ca+ +-mobilizing agonists increases basal intracellular pH in hepatocytes. To explain this difference in effects of agents that increase intracellular Ca+ + concentration, the mechanism of ionomycin-induced increases in basal intracellular pH in isolated rat hepatocytes was studied. Changes in intracellular pH and intracellular Ca+ + concentration were measured with the fluorescent probes BCECF (2′,7′-bis-2-[carboxyethyl ester]-5carboxyfluorescein) and quin-2, respectively. Ionomycin produced dose-dependent increases in intracellular pH and intracellular Ca+ + concentration, with the increase in intracellular Ca+ + concentration preceded by the increase in intracellular pH. Ionomycin-induced increases in intracellular pH were not affected by I mmol/L amiloride, 100 μmol/L diisothiocyanostilbene disulfonate or removal of extracellular Na+, indicating that the effect is not mediated by Na+/H+ exchange, Cl−/HCO3− exchange or Na+/HCO3− cotransport. Ionomycin failed to increase intracellular pH or intracellular Ca+ + concentration in the absence of extracellular Ca+ +, and both intracellular pH and intracellular Ca+ + concentration increased promptly when extracellular Ca+ + was reintroduced. Ionomycin-induced increases in intracellular Ca+ + concentration but not intracellular pH were smaller in hepatocytes loaded with the Ca+ + buffering agent MAPTA. Thapsigargin increased intracellular Ca+ + concentration but failed to increase intracellular pH. Thus the effect of ionomycin is independent of the effect of ionomycin on intracellular Ca+ + concentration and dependent on extracellular intracellular Ca+ + concentration. Experimental conditions that produce cell depolarization did not increase basal intracellular pH but lowered ionomycininduced increases in intracellular pH by 25% without affecting increases in intracellular Ca+ + concentration. Taken together, these results indicate that the increase in basal intracellular pH may primarily be due to ionomycin-mediated electroneutral Ca+ +/2H+ exchange across the hepatocyte plasma membrane. Because the effect of ionomycin is not mediated by Na+/H+ exchange, the activity of this exchanger under basal conditions is not regulated by intracellular Ca+ +. These results also suggest that the pharmacological effects of ionomycin in hepatocytes are mediated by changes in intracellular pH in addition to or independent of changes in intracellular Ca+ + concentration. (HEPATOLOGY 1993;18:433–439).