Fluctuations of intracellular Ca2+ ([Ca2+]i) regulate a variety of cellular functions. The classical Ca2+ transport pathways in the endoplasmic reticulum (ER) and plasma membrane are essential to [Ca2+]i oscillations. Although mitochondria have recently been shown to absorb and release Ca2+ during G protein-coupled receptor (GPCR) activation, the role of mitochondria in [Ca2+]i oscillations remains to be elucidated. Using fluo-3-loaded human teratocarcinoma NT2 cells, we investigated the regulation of [Ca2+]i oscillations by mitochondria. Both the muscarinic GPCR agonist carbachol and the ER Ca2+-adenosine triphosphate inhibitor thapsigargin (Tg) induced [Ca2+]i oscillations in NT2 cells. The [Ca2+]i oscillations induced by carbachol were unsynchronized among individual NT2 cells; in contrast, Tg-induced oscillations were synchronized. Inhibition of mitochondrial functions with either mitochondrial blockers or depletion of mitochondrial DNA eliminated carbachol--but not Tg-induced [Ca2+]i oscillations. Furthermore, carbachol-induced [Ca2+]i oscillations were partially restored to mitochondrial DNA-depleted NT2 cells by introduction of exogenous mitochondria. Treatment of NT2 cells with gap junction blockers prevented Tg-induced but not carbachol-induced [Ca2+]i oscillations. These data suggest that the distinct patterns of [Ca2+]i oscillations induced by GPCR and Tg are differentially modulated by mitochondria and gap junctions.