Changes in intracellular calcium concentration play a major role both in signal transduction and in cell death. In particular, mitochondrial Ca2+ overload is critically important as a determinant of irreversible cell injury. When accumulated above a threshold, matrix Ca2+ triggers opening of the mitochondrial permeability transition pore (mPTP), initiating ATP depletion and cell death via necrosis or by promoting cytochrome c release and initiating the apoptotic cascade. Measurement of mitochondrial Ca2+ uptake capacity (or the threshold for mPTP opening) is, therefore, important for understanding the mechanisms of pathophysiology in a variety of disease models and also for testing neuro- or cardioprotective drugs. We have, therefore, devised an approach that delivers Ca2+ directly to the matrix of mitochondria independently of uptake and therefore independently of potential (Δψm) that allows direct study both of the Ca2+ efflux pathway and of the specific sensitivity of mPTP to Ca2+. This is achieved using the photolytic release of Ca2+ by flash photolysis of caged Ca2+ using compounds, such as o-nitrophenyl EGTA, introduced into the cell as the acetoxymethyl (AM) ester (NP-EGTA, AM). This method can be used in both intact and permeabilized cells.