The distribution of pyruvate, phosphate, malate, citrate, K+, aspartate, glutamate, ADP, and ATP between the mitochondrial and cytosolic compartments was studied in isolated rat hepatocytes exposed to 30 min anoxia. The results show that pyruvate and citrate gradients are comparable to aerobic values, indicating that the pH gradient across the membrane under anaerobic conditions is comparable to that under normal aerobic conditions. In contrast, the distribution of phosphate, malate, ATP, ADP, aspartate, and glutamate suggests that transport of these species may be inhibited during anoxia. The results are discussed in terms of potential regulation of mitochondrial function to provide a quiescent anoxic state that is capable of recovering normal function on reoxygenation.