The endogenous reactive oxygen species ( ROS ) formation is associated with many pathologic states such as inflammatory diseases, neurodegenerative diseases, brain and heart ischemic injuries, cancer, and aging. The purpose of this study was to investigate the endogenous sources for ROS formation in intact isolated rat hepatocytes, in particular, peroxisomal oxidases, monoamine oxidase, xanthine oxidase, cytochrome P450, and mitochondria electron transport. The rat hepatocyte cat alyzed oxidation of 2’,7’- dichlorofluorescin to form the fluorescent 2,7’-dichlorofluorescein was used to measure endogenous and xenobiotic-induced reactive oxygen speci es ( ROS ) formation by intactisolated rat hepatocytes. Various oxidase substrates and inhibitors were then used to identify the intracellular oxidases responsible. Endogenous ROS formation was markedly increased in catalase inhibited or GSH depleted hepatocytes, and was inhibited by ROS scavengers or des feroxamine. Endogenous ROS formation was also inhibited by cytochrome P450 inhibitors, but was not affected by oxypurinol, a xanthine oxidase inhibitor. Mitochondrial respiratory chain inhibitors or hypoxia, on the other hand, markedly increased ROS before cytotoxicity ensued. This suggests endogenous ROS formation can largely be attributed to oxygen reduction by reduced mitochondrial el ectron transport components and reducedcytochrome P450 isozymes. Addition of monoamine oxidase substrat es increased antimycin Aresistant respiration and ROS formation before cytotoxicity ensued. On the other hand peroxisomal substrates readily induced ROS formation and were cytotoxic towards catalaseinhibited hepatocytes, which suggests that peroxisomal catalase removes endogenous H2O2 formed in the peroxisomes. The consequences of upregulation of peroxisomal oxidases are discussed.