STUDY OBJECTIVE - The aim of the study was to measure variations in ATP synthase capacity in cultured cardiomyocytes under conditions of metabolic stimulation. DESIGN - ATP synthase activity was measured in cultured rat cardiomyocytes using a procedure which allowed rapid measurement of mitochondrial function during changes in metabolic state. EXPERIMENTAL MATERIAL - Calcium tolerant cardiomyocytes were prepared from male Wistar rats, weight 250-300 g, n = 6-22 per experiment. MEASUREMENTS AND MAIN RESULTS - Electrical stimulation of cardiomyocytes led to an approximate doubling of ATP synthase capacity within 1-2 min, and was rapidly reversible. Activation was reduced when extracellular calcium was lowered and abolished in presence of the calcium entry blocker ruthenium red. Exposure of cardiomyocytes to isoprenaline or to an inhibitor of phosphodiesterase III also led to a large increase in ATP synthase capacity, which was abolished in presence of ruthenium red. However, the response of cells to isoprenaline depended on their pretreatment: activation of ATP synthase was abolished after 20 min anoxia prior to isoprenaline treatment but regained after a subsequent 30 min reoxygenation. This may reflect down regulation of beta receptors on the cell surface during anoxia. CONCLUSIONS - ATP synthase is directly controlled in vivo by a non-allosteric mechanism. Activation of ATP synthase is a response to intramitochondrial Ca2+ concentration.