An increase in intracellular Ca2+ ([Ca2+]i) is necessary for mast cell exocytosis, but there is controversy over the requirement for Ca2+ in the extracellular medium. Here, we demonstrate that mitochondrial function is a critical determinant of Ca2+ dependence. In the presence of extracellular Ca2+, mitochondrial metabolic inhibitors, including rotenone, antimycin A, and the protonophore carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP), significantly reduced degranulation induced by immunoglobulin E (IgE) antigen or by thapsigargin, as measured by beta-hexosaminidase release. In the absence of extracellular Ca2+; however, antimycin A and FCCP, but not rotenone, enhanced, rather than reduced, degranulation to a maximum of 76% of that observed in the presence of extracellular Ca2+. This enhancement of extracellular, Ca2+-independent degranulation was concomitant with a rapid collapse of the mitochondrial transmembrane potential. Mitochondrial depolarization did not enhance degranulation induced by thapsigargin, irrespective of the presence or absence of extracellular Ca2+. IgE antigen was more effective than thapsigargin as an inducer of [Ca2+]i release, and mitochondrial depolarization augmented IgE-mediated but not thapsigargin-induced Ca2+ store release and mitochondrial Ca2+ ([Ca2+]m) release. Finally, atractyloside and bongkrekic acid [an agonist and an antagonist, respectively, of the mitochondrial permeability transition pore (mPTP)], respectively, augmented and reduced IgE-mediated Ca2+ store release, [Ca2+]m release, and/or degranulation, whereas they had no effects on thapsigargin-induced Ca2+ store release. These data suggest that the mPTP is involved in the regulation of Ca2+ signaling, thereby affecting the mode of mast cell degranulation. This finding may shed light on a new role for mitochondria in the regulation of mast cell activation.