Mitochondrial involvement in Ca2+ signaling is thought to be due to the effect of mitochondrial Ca2+ removal from and Ca2+ release to cytosolic domains close to ryanodine and IP3 Ca2+ channels. However, mitochondria are a source of low levels of endogenous reactive oxygen species, and Ca2+ release channels are known to be redox-sensitive. In the present work, we studied the role of mitochondrial production of oxygen species in Ca2+ oscillations during physiological stimulation. Mitochondria-targeted antioxidants and mitochondrial inhibitors quickly inhibited calcium oscillations in pancreatic acinar cells stimulated by postprandial levels of the gut hormone cholecystokinin. Confocal microscopy using different redox-sensitive dyes showed that cholecystokinin-induced oscillations are associated with mitochondrial production of reactive oxygen species. This production is inhibited by application of mitochondria-targeted antioxidants and mitochondrial inhibitors. In addition, we found no correlation between inhibition of oscillations and mitochondrial depolarization. We conclude that low level production of reactive oxygen species by mitochondria is a necessary element in the development of Ca2+ oscillations during physiological stimulation. This study unveils a new and unexplored aspect of the participation of mitochondria in calcium signals. J. Cell. Physiol. 206: 487–494, 2006. © 2005 Wiley-Liss, Inc.