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Here, we report that choline and dimethylglycine can stimulate reactive oxygen species (ROS) production in liver mitochondria. Choline stimulated O ˙ /H O formation at a concentration of 5 μm. We also observed that Complex II and III inhibitors, atpenin A5 and myxothiazol, collectively induced a 95% decrease in O ˙ /H O production indicating both sites serve as the main sources of ROS during choline oxidation. Dimethylglycine, an intermediate of choline oxidation, was a more effective ROS generator. Rates of production were ~ 43% higher than choline-mediated O ˙ /H O production. The main site for dimethylglycine-mediated ROS production was via reverse electron transfer to Complex I. Our results demonstrate that metabolism of essential metabolites involved in methionine and folic acid biosynthesis can stimulate mitochondrial ROS production.