Abstract: Melatonin is a potent scavenger of reactive oxygen (ROS) and reactive nitrogen species (RNS). At pharmacological concentrations, however, melatonin is documented to cause ROS/RNS production, especially in cultured cancerous cells. Currently, the mechanism responsible for melatonin-induced ROS generation remains elusive. In this study, we provided evidence that melatonin, at micromolar concentrations, induced rapid ROS generation by a mitochondrial-dependent mechanism in primary human mesangial (HM) cells. The melatonin-induced ROS production occurred independent of changes in Ca2+ concentrations in the cytosol and/or in mitochondria. In mitochondria isolated from HM cells and mice kidney tissues, melatonin caused ROS production; this melatonin response was completely blocked by the complex III inhibitor antimycin A. In contrast, both the mitochondrial complex I inhibitor, rotenone, and another complex III inhibitor, myxothiazol, which interacts with complex III at a distinct site, had no significant inhibitory effect on melatonin-induced ROS generation. These results demonstrate that melatonin induced rapid ROS generation via the antimycin A-sensitive site of mitochondrial complex III.