The formation of oxygen reactive species in response to oxidative stimuli was measured in rat synaptosomes. Studies employed the non-fluorescent probe 2?,7?-dichlorofluorescin diacetate (DCFH-DA), which after de-esterification is oxidized in the presence of oxygen reactive species to the highly fluorescent 2?,7?-dichlorofluorescein (DCF). Oxygen reactive species formation, as measured by DCF fluorescence, was stimulated by ascorbate and/or FeSO(4), and xanthine/xanthine oxidase under various buffering conditions. These agents all increased DCF formation in Tris, HEPES and phosphate buffer. Ascorbate also stimulated the formation of DCF in a concentration-dependent manner. The presence of Ca(2+) in HEPES buffer did not enhance or diminish the effects of ascorbate/FeSO(4) on DCF formation. Deferoxamine inhibited the ascorbate/FeSO(4)-induced stimulation of DCF formation, but xanthine/xanthine oxidase-induced stimulation was not affected by pretreatment with superoxide dismutase. Results indicate that DCF fluorescence is a sensitive, quantitative and direct measure of oxygen reactive species formation in synaptosomes, providing a rapid method for investigating early neuronal events that occur during oxidative stress.