The influence of cytosolic Ca(2+) load and of mitochondrial membrane potential change on mitochondrial morphology was investigated in cultured rat hippocampal astrocytes. The uncoupler FCCP, applied together with oligomycin, depolarized mitochondria rapidly but did not change their morphology. Depolarization was associated with a moderate cytosolic [Ca(2+)](i) rise of up to 0.3 microM. Only high cytosolic Ca(2+) load (above a threshold of 50 microM), which was evoked by application of the ionophore 4-Br-A23187 in Ca(2+)-containing medium, caused drastic change of mitochondrial morphology. The shape change from the typical rod-like to a spherical shape, indicating mitochondrial swelling, was associated with depolarization. Cyclosporin A sensitivity suggests involvement of permeability transition. Thus, a dramatic cytosolic [Ca(2+)](i) rise is required to induce mitochondrial swelling and depolarization. A large but still moderate [Ca(2+)](i) rise evoked by physiological stimulation, however, has no comparable effect.