The c-Jun N-terminal kinases (JNKs) are a subfamily of mitogen-activated protein kinases, when activated in response to stresses such as ischemia and reperfusion, might pathologically phosphorylate various substrates such as c-Jun and ATF2. JNKs are encoded by three genes Jnk1, Jnk2, and Jnk3 to form up to 10 spliceforms and strikingly Jnk3-/- animals have shown promising neuroprotection against stroke. Several JNK inhibitors, including a JNK peptide inhibitor TAT-TIJIP that acts as JNK binding domain (JBD) competitor, have been developed to study the functions of JNKs, and can prevent neuronal death following excitatory glutamate insult. To address the outstanding issue of the JNK partners or substrates that may mediate this excitotoxicity, this study has employed the yeast two-hybrid approach to discover new binding partners for both JNK1 and JNK3. Further mutagenesis of the JNK1 substrate binding groove combined with testing for interactions in the yeast two-hybrid system has allowed the definition of these interacting partners as either typical, JBD-dependent interactors, or atypical JNK interactors. Bioinformatics predictions of putative JBDs in the JNK1 and JNK3 interactors is now directing analyses to define the protein-protein interaction interfaces that drive high affinity interaction with JNK1 and/or JNK3. Thus, our identification and characterization of the JNK1/JNK3 interactome of the brain will drive further evaluation of the contributions by the JNKs to excitotoxicity.