&NA;We investigated the mechanisms by which spontaneously beating cultured rat ventricular cells regulate intracellular pH (pHi). Specifically, the relative contributions of the Na+/H+antiport, Cl−/HCO3−exchange, ATP, and calmodulin‐dependent processes in regulating the pHiof cells loaded with the intracellular fluorescent pH indicator BCECF were investigated. The pHiof ventricular cells bathed in HEPES‐buffered medium averaged 7.30±0.02. Subsequent exposure of the cells to CO2‐HCO3−‐buffered medium resulted in intracellular acidification followed by recovery to pHilevels approximately 0.1 pH units lower than in controls. Recovery was inhibited by the Na+/H+antiport inhibitor 5‐(N‐ethyl‐N‐isopropyl) amiloride (EIPA). The recovery from intracellular acidification, induced by a 15‐mM ammonium chloride prepulse, was also dependent solely upon activation of the Na+/H+antiport. Recovery was dependent upon extracellular sodium, was completely inhibited by EIPA, and could be modulated by changes in extracellular pH (pHo). At low pHovalues (6.3) the recovery of pHiwas greatly attenuated, while at high pHo(8.0) the recovery process was accelerated. The final pHito which the cells recovered was also dependent upon pHo. Preincubation of the cells with 2‐deoxy‐D‐glucose to deplete cellular ATP levels reduced pHiby approximately 0.2 pH units and greatly impaired the cells' ability to recover from 15‐mM ammonium chloride‐induced acid load. Similarly, preincubation of cells with the calmodulin inhibitors W‐7 and trifluoperazine also impaired their ability to recover from the acid load. The Cl−‐HCO3−exchange played no role in the cells' ability to recover from intracellular acidosis. However, the presence of HCO3−significantly increased the resistance of myocardial cells to changes in pHiby approximately doubling their buffer capacity. These results demonstrated that a Na+/H+antiport is the major pHi‐regulating system in spontaneously beating rat ventricular cells. The ability of the Na+/H+antiport to regulate myocardial pHiis dependent upon the cells' ability to maintain adequate levels of ATP. The antiport's dependency on ATP, in conjunction with its dependency on calmodulin, suggests that activation of the antiport in ventricular cells involves phosphorylation processes. (Circulation Research1989;64:676‐685)