Oxidative stress activates the c-Jun N-terminal kinase (JNK) pathway. However, the exact mechanisms by which reactive oxygen species (ROS) activate JNK are unclear. We found that the ability of hydrogen peroxide (H 2 O 2) to induce JNK activation varied in different cell types. Pyrrolidine dithiocarbamate (PDTC), a presumed antioxidant, induced JNK activation on its own and enhanced JNK activation by H 2 O 2 in many cell types, including Jurkat, HEK293, and LNCaP and Tsu-Pr1 prostate cancer cells. The activation of JNK by PDTC, in the presence or absence of exogenous H 2 O 2, was dependent on its chelating ability to metal ions, most likely copper ions. Despite the strong JNK-activating ability, H 2 O 2 plus PDTC did not induce significant activation of the upstream kinases, SEK1/MKK4 and MKK7. However, the JNK inactivation rate was slower in cells treated with H 2 O 2 plus PDTC compared with the rate in cells treated with ultraviolet C (UV-C). Treatment of H 2 O 2 plus PDTC significantly decreased the expression levels of a JNK phosphatase, M3/6 (also named hVH-5), but not the levels of other phosphatases (PP2A and PP4). In contrast, UV-C irradiation did not cause the down-regulation of M3/6. These results suggest that JNK activation by H 2 O 2 plus PDTC resulted from the down-regulation of JNK phosphatases. Our data also reveal a necessity to carefully evaluate the pharmacological and biochemical properties of PDTC.