Reactive oxygen species (ROS) released from (dys-)functioning mitochondria contribute to normal and pathophysiological cellular signaling by modulating cytosolic redox state and redox-sensitive proteins. To identify putative redox-targets involved in such signaling we exposed hippocampal neurons to H₂O₂. Redox-sensitive dyes indicated that external H₂O₂ may oxidize intracellular targets in cultures and slices. In cultured neurons H₂O₂ (EC₅₀ 100 mM) induced an intracellular Ca²⁺ rise which persisted upon Ca²⁺ withdrawal and mitochondrial uncoupling. It was, however, antagonized by thapsigargin, high levels of ryanodine, and dantrolene, which identifies the endoplasmic reticulum (ER) as the Ca²⁺ store involved. Intracellular accumulation of endogenously generated H₂O₂– provoked by inhibiting glutathione peroxidase – also released Ca²⁺ from the ER. Extracellular generation of superoxide increased the intracellular Ca²⁺ level as well. Phospholipase C mediated metabotropic signaling was depressed in the presence of H₂O₂, but cytosolic cAMP levels were not affected. H₂O₂ (1–5 mM) moderately depolarized mitochondria, halted their intracellular trafficking in a cAMP-independent manner, and directly oxidized cellular NADH and FADH₂. In part, the mitochondrial depolarization reflects uptake of Ca²⁺ previously released from the ER. We conclude that H₂O₂ releases Ca²⁺ from the ER via ryanodine receptors; mitochondrial function is not markedly impaired. Such modulation of Ca²⁺ signaling and organelle interaction by ROS affects the efficacy of metabotropic signaling cascades and may contribute to the adjustment of neuronal function to oxidative and/or metabolic disturbances.