In the present work, the Ca2+ dependence of mitochondrial H2O2 elimination was investigated. Mitochondria isolated from guinea pig brain were energized by glutamate and malate and incubated with micromolar concentrations of Ca2+ in the presence of ADP, preventing permeability transition pore formation. After the completion of Ca2+ uptake, mitochondria were challenged with H2O2 (5 μM), then at various time points residual H2O2 was determined using the Amplex red method and compared with that in mitochondria incubated with H2O2 without Ca2+ addition. Dose-dependent inhibition of H2O2 elimination by Ca2+ was detected, which was prevented by the Ca2+-uptake inhibitor Ru 360. Stimulation of Ca2+ release from Ca2+ -loaded mitochondria by a combined addition of Ru 360 and Na+ decreased the Ca2+-evoked inhibition of H2O2 removal. After Ca2+ uptake (50 μM), mitochondrial aconitase activity was found to be decreased, which was partially attributable to the impaired elimination of endogenously produced reactive oxygen species. We found that the effects of Ca2+ and H2O2 on the activity of aconitase were additive. These results confirm that Ca2+ inhibits elimination of H2O2 in mitochondria and demonstrate that this effect is concentration dependent and reversible. The phenomenon described here can play a role in the modulation of ROS handling under conditions involving excessive cellular Ca2+ load. © 2011 Wiley-Liss, Inc.