Multiple myeloma (MM) is a presently incurable B-cell malignancy, and newer biologically based therapies are needed. Arsenic trioxide (ATO) has been established as a therapeutic agent for relapsed acute promyelocytic leukemia patients, and has been used for MM patients in clinical trials. In this study, we investigated the role of c-jun-N-terminal kinase (JNK) in ATO-induced apoptosis in MM lines. The exogenous interleukin (IL)-6 dependent MM line, ILKM-3, and independent MM lines, U266 and XG-7, were treated with a therapeutic concentration of ATO with or without JNK inhibitor 1 (a JNK-specific inhibitor) and anisomycin (a JNK activator). Their cell growth, cell cycle, JNK activation and NF-kappaB activation were investigated. ATO induced apoptosis in U266 and ILKM-3 regardless of their exogenous IL-6 dependency. This apoptosis, accompanied with decreased mitochondrial transmembrane potential, sustained activation of JNK but not cell cycle arrest. Pretreatment of JNK inhibitor prevented ATO-induced apoptosis in ATO-sensitive lines. Combined treatment with ATO and anisomycin induced sustained activation of JNK and apoptosis in the ATO-insensitive MM line, XG-7. Results of various time period treatments of ATO showed that sustained activation of JNK was needed in ATO-induced apoptosis in MM. IkBalpha phosphorylation was not associated with ATO-sensitivity of MM lines. These findings suggest that sustained activation of JNK plays a critical role in ATO-induced apoptosis in MM cell lines. Cotreatment with ATO and the agent, which can induce sustained activation of JNK, might improve the outcome in MM therapy.