An efficient and reliable method for measuring ROS production in intact functional mitochondria is described. The method is specific for H2O2 and is sensitive enough to assay basal H2O2 production in the respiratory chain. It shows high reproducibility, generates stable final fluorescence, and does not interfere with mitochondrial function. The assay system shows instantaneous response to H2O2, and is thus useful for studying the effect of inhibitors or modulators on the rate of ROS production. It also allows the calculation of the free radical leak (the percentage of electrons out of sequence which reduce O2 to ROS instead of to water at the respiratory chain). While other fluorescent methods are available to measure

ROS production in isolated mitochondria, the one presented here has various advantages: it is based on increases (instead of decreases) in fluorescence, reactants are not expensive, and homovanillic acid does not spontaneously generate fluorescence. The method, similarly to the available alternatives, can be applied only to isolated mitochondria. The measurement of ROS production (independent of ROS scavenging) in intact cells or animals must wait for future technical developments.

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