CCl4 rapidly and severely inhibits hepatic endoplasmic reticulum calcium (Ca++) sequestration in rats exposed to this hepatotoxin in vivo. As a consequence, it is possible that cytosolic Ca++ concentrations become elevated in liver cells. In this study, the authors examined intracellular Ca++ concentrations in cultured rat hepatocytes exposed to CCl4 by monitoring the activity of phosphorylase a. Glycogen phosphorylase is converted to its a form in response to increases in cytoplasmic Ca++. Elevated phosphorylase a activity was observed within 2.5 min and was maintained for at least 30 min after exposure of hepatocytes to CCl4. Endoplasmic reticulum Ca++ pump activity decreased in a parallel manner. Phosphorylase activation was cyclic AMP independent and did not require extracellular Ca++. Cytoplasmic enzyme was released from hepatocytes within 30 min after CCl4 addition. Thus it was confirmed that exposure of hepatocytes to CCl4 causes release of Ca++ from an intracellular store (likely endoplasmic reticulum) and resultant activation of a Ca++-responsive cytosolic enzyme. From a calibration curve, it was estimated that cytosolic Ca++ is elevated up to 100-fold in rat hepatocytes exposed to the model hepatotoxin CCl4. It is postulated that prolonged elevation of intracellular Ca++ concentrations may trigger excessive stimulation of Ca++-sensitive enzymes capable of initiating irreversible liver cell injury.