Abstract C-Jun N-terminal kinases (JNKs) regulate various cellular functions. Their activation is mediated cooperatively by both MKK4 and MKK7. The c-Jun N-terminal kinase 2 (JNK2) isoform is constitutively activated in glial tumor cell lines and human glioblastoma models. We investigated the regulation of JNK2 self-activation in vitro in both cell-free and cell-based experiments. Light scattering analysis suggested that unphosphorylated recombinant JNK2α2 exists in solution as a mixture of monomers, dimers and tetramers. JNK2α2 self-phosphorylates more rapidly in vitro when assayed at low concentration leading to its activation. However, at a slightly higher concentration, the formation of a tetramer is favored, whose ability to self-phosphorylate is suppressed. HEK293T cells that were transfected with varying amounts of pcDNA-JNK2α2 showed that an increased concentration of JNK2α2 (54 kDa) could suppress the appearance of activated JNK2α2 in mammalian cells. A mutant of JNK2 (F170R) thought to stabilize the DFG-out conformation appears to stabilize the formation of a JNK2 tetramer. Taken together our data suggest that oligomerization may regulate non-canonical (self-activation) activation of JNK2. Citation Format: Tamer S. Kaoud, Austin F. Riggs, Kevin N. Dalby. JNK2 oligomerization regulates its activation through non-canonical pathways. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3227. doi:10.1158/1538-7445.AM2014-3227