Cysteine oxidase, which catalyzes the oxidation of cysteine to the neuroexcitatory and neurotoxic compounds cysteine sulfinic and sulfonic acids, previously has not been studied successfully in brain. Here we report optimal conditions for measuring cysteine oxidase activity in rat brain, describe its regional and subcellular distribution and compare activity during several stages of development. Employing NAD+ as cofactor, substantial activity was measurable in whole brain homogenate, 70% of which was localized to the microsomal fraction. Appreciable activity was present throughout brain with a 3-fold difference being detected between pons-medulla and cerebral cortex, the regions with the highest and lowest levels respectively. Cysteine oxidase activity in adult rat brain was approximately 5 times higher than that measured at birth. For the first 6 days of life there was very little change in activity; thereafter, a steady increase occurred with adult levels being reached between the 25th and 50th days.