Pancreatic -cells are susceptible to reactive oxygen species (ROS), which are known to be generated by high or low glucose, hypoxic, or cytokine-producing conditions. When we cultured mouse -cell-derived MIN6 cells in a low glucose condition, we detected a significant generation of ROS, including hydrogen peroxide, which was comparable to the ROS production in hypoxic or cytokine-treated conditions. ROS accumulation induced by the low glucose culture led to cell death, which was prevented by the ROS scavengers N-acetylcysteine and MnTBAP. We next investigated the mechanism of stress-activated protein kinases (SAPKs), JNK and p38, in ROS-induced MIN6 cell death. Activation of p38 occurred immediately after the low glucose culture, whereas JNK activation increased slowly 8 h later. Adenoviral p38 expression decreased MIN6 cell death whereas the JNK expression increased it. Consistently, blocking p38 activation by inhibitors increased -cell death, whereas JNK inhibitors decreased it. We then examined the role of MAP kinase phosphatases (MKPs) specific for SAPKs in -cell death. We found that MKP-1 presented an increase in its oxidized product after the low glucose culture. ROS scavengers prevented the appearance of this oxidized product and JNK activation. Thus, ROS-induced MKP inactivation causes sustained activation of JNK, which contributes to -cell death. Adenoviral overexpression of MKP-1 and MKP-7 prevented the phosphorylation of JNK at 36 h after the low glucose culture and decreased MIN6 -cell death. We suggest that -cell death is regulated by interactions between JNK and its specific MKPs.