Release of Ca2+ from mitochondria into cytosol in intact thymocytes was studied using the fluorescent dye Fluo-3. It was shown that the release of Ca2+ induced by the dithiol cross-linking agent phenylarsine oxide or by uncoupler was strongly inhibited by cyclosporin A, a specific inhibitor of the permeability transition pore (PTP) in mitochondria. Oxidative stress sensitized the pore so even partial uncoupling caused rapid cyclosporin A-sensitive release of Ca2+. The experiments on digitonin-permeabilized cells confirmed that uncoupling induced opening of the PTP, which forms the major pathway for rapid release of Ca2+ from thymocyte mitochondria.