Ca2+ release from mitochondria can be induced by a variety of chemically different prooxidants. Release induced by these compounds is possibly regulated by protein mono(ADP)ribosylation, and leaves mitochondria initially intact. Excessive "cycling" (continuous release and uptake) of Ca2+ by mitochondria leads to their damage, as shown by a decreased membrane potential, fast Ca2+ release, and impairment of ATP synthesis. When cycling is prevented by Ca2+ chelators or by inhibition of the uptake route with ruthenium red, prooxidants still induce Ca2+ release but mitochondria remain intact. It has recently been suggested that formation of a "pore" in the inner mitochondrial membrane participates in the Ca2+ release mechanism. We find that the prooxidant-induced Ca2+ release is not paralleled by sucrose entry into, or K+ release from, or swelling of mitochondria, provided Ca2+ cycling is prevented. Thus, the prooxidant-induced Ca2+ release does not require formation of a "pore." We conclude that the release occurs via a specific pathway.