We have previously shown that Ang II induces mesangial cell (MC) proliferation via JNK-AP-1 pathway. The present study attempted to determine the upstream mediators of the JNK activation with emphasis on reactive oxygen species (ROS) and the epidermal growth factor receptor (EGFR). In cultured human mesangial cells (HMCs), Ang II time-dependently increased intracellular ROS production that was sensitive to diphenyleneiodonium sulfate (10 µM) and apocynin (500 µM), two structurally distinct NADPH oxidase inhibitors. In contrast, inhibitors of other oxidant-producing enzymes, including the mitochondrial complex I inhibitor rotenone (ROT), the xanthine oxidase inhibitor allopurinol, the cyclooxygenase inhibitor indomethacin, the lipoxygenase inhibitor nordihydroguiaretic acid, the cytochrome P450 oxygenase inhibitor ketoconazole and the nitric oxide synthase inhibitor G-nitro-L-arginine methyl ester (L-NAME) were without an effect. The increased ROS production was associated with translocation of cytosolic p47^phox and p67^phox to the membrane as assessed by immunoblotting analysis of fractionated cellular proteins. The antioxidants almost abolished Ang II-mitogenic response, as assessed by [³H]thymidine incorporation and cell number counting, associated with a remarkable blockade of the activation of EGFR and JNK. The EGFR inhibitor AG1478 was able to mimic the effect of antioxidants in inhibiting the mitogenic response and the JNK activation following Ang II treatment. Together, these data suggest ROS/EGFR/JNK pathway in transducing the proliferative effect of Ang II in cultured HMCs.