Although many cell functions are regulated by Ca2+ oscillations induced by a cyclic release of Ca2+ from intracellular Ca2+ stores, the pacemaker mechanism of Ca2+ oscillations remains to be explained. Using green fluorescent protein-based Ca2+ indicators that are targeted to intracellular Ca2+ stores, the endoplasmic reticulum (ER) and mitochondria, we found that Ca2+ shuttles between the ER and mitochondria in phase with Ca2+ oscillations. Following agonist stimulation, Ca2+ release from the ER generated the first Ca2+ oscillation and loaded mitochondria with Ca2+. Before the second Ca2+ oscillation, Ca2+ release from the mitochondria by means of the Na+/Ca2+ exchanger caused a gradual increase in cytoplasmic Ca2+ concentration, inducing a regenerative ER Ca2+ release, which generated the peak of Ca2+ oscillation and partially reloaded the mitochondria. This sequence of events was repeated until mitochondrial Ca2+ was depleted. Thus, Ca2+ shuttling between the ER and mitochondria may have a pacemaker role in the generation of Ca2+ oscillations.