c-Jun N-terminal kinases (JNKs) are members of the mitogen-activated protein kinase (MAPK) family that regulate apoptosis, inflammation, cytokine production, and metabolism. MAPKs undergo various splicing within their kinase domains. Unlike other MAPKs, JNKs have alternative splicing at the C-terminus, resulting in long and short variants. Functional or conformational effects due to the elongated C-terminal tail in the long splice variants have not been investigated nor has the conformation of the C-terminal tail been analyzed. Here, we analyzed the conformation of the elongated C-terminal tail and investigated conformational differences between long and short splice variants of JNKs using JNK3 alpha 2 and JNK3 alpha 1 as models. We adopted hydrogen/deuterium exchange mass spectrometry (HDX-MS) to analyze the conformation. HDX-MS revealed that the C-terminal tail is mostly intrinsically disordered, and that the conformation of the kinase domain of JNK3 alpha is more dynamic than that of JNK3 alpha 1. The different conformation dynamics between long and short splice variants of JNK3 alpha might affect the cellular functions of JNIG. (C) 2016 Elsevier Inc. All rights reserved.