It is well established that menadione, 2-methyl-l,4-naphthoquinone, impairs the ability of rat liver mitochondria to accumulate and retain calcium. However, it remains unclear whether this reflects inhibition of mitochondrial calcium uptake or stimulation of calcium release by menadione. The purpose of the current investigation was to determine whether interference with mitochondrial calcium homeostasis by menadione reflects a selective activation of the cyclosporine A-sensitive pore, independent of actions on other mitochondrial calcium channels. Mitochondrial calcium flux was monitored using the metal- lochromic dye arsenazo III. Treatment of mitochondria with menadione caused a concentration-dependent decrease in net calcium accumulation followed by a delayed release of the accumulated calcium and concurrent mitochondrial swelling. Both the maximum steady-state accumulation of calcium and the delay preceding calcium release decreased as a function of calcium concentration. The release of calcium did not occur via the Na+/Ca2+antiport or reversal of the uptake uniport, as neither diltiazem nor ruthenium red prevented the menadione-stimulated calcium release. In contrast, cyclosporine A, a potent inhibitor of the permeability transition pore, completely inhibited menadione-induced calcium release and the associated swelling. Furthermore, the menadione-induced inhibition of calcium accumulation was completely prevented in the presence of cyclosporine A, indicating a selective stimulation of calcium release by menadione, rather than inhibition of calcium uptake. These data provide the first definitive description of a specific action of menadione to stimulate mitochondrial calcium release through a cyclosporine A-sensitive pathway, independent of altering the regulation of other recognized calcium channels associated with the inner mitochondrial membrane.