WR1065 is an aminothiol with selective cytoprotective effects in normal cells compared with cancer cells. In a previous study (North, S., El-Ghissassi, F., Pluquet, O., Verhaegh, G., and Hainaut, P. (2000) Oncogene 19, 1206-1214), we have shown that WR1065 activates wild-type p53 in cultured cells. Here we show that WR1065 induces p53 to accumulate through escape from proteasome-dependent degradation. This accumulation is not prevented by inhibitors of phosphatidylinositol 3-kinases and is not accompanied by phosphorylation of Ser-15, -20, or -37, which are common targets of the kinases activated in response to DNA damage. Furthermore, WR1065 activates the JNK (c-Jun N-terminal kinase), decreases complex formation between p53 and inactive JNK, and phosphorylates p53 at Thr-81, a known site of phosphorylation by JNK. A dominant negative form of JNK (JNK-APF) reduces by 50% the activation of p53 by WR1065. Thus, WR1065 activates p53 through a JNK-dependent signaling pathway. This pathway may prove useful for pharmacological modulation of p53 activity through non-genotoxic mechanisms.