Ca2+/calmodulin-dependent protein kinase IV (CaM-KIV) is thought to be involved in regulating gene expression by phosphorylating various transcriptional factors. CaM-KIV as well as CaM-KI are activated upon phosphorylation by two distinct isoforms of Ca2+/calmodulin-dependent protein kinase kinases, CaM-KKs α and β. In this study, we raised isoform-specific monoclonal antibodies against CaM-KKs and examined the immunohistochemical localization of CaM-KKs in the rat brain, compared with that of CaM-KIV. CaM-KK α-immunoreactivity was rather widely distributed in neurons throughout the brain, except cerebellar cortex. The highest levels of CaM-KK α-immunoreactivity were observed in the cerebral cortex, facial nucleus and motor neurons of the spinal cord. Moderate CaM-KK α-immunoreactivity was observed in the hippocampal formation, pontine nuclei and various brain stem nuclei including trigeminal, vestibular, cochlear and hypoglossal nuclei. In contrast, CaM-KK β-immunoreactivity was relatively restricted in some neuronal populations. The highest levels of CaM-KK β-immunoreactivity were observed in the cerebellar granule cell layer, and moderate immunoreactivity was observed in the cerebral cortex, hippocampal formation, caudate putamen, pontine nuclei, cochlear nucleus and molecular layer of the cerebellum. In contrast to the prominent nuclear localization of CaM-KIV, both isoforms of CaM-KKs were localized in the perikaryal cytoplasm, dendrites and nerve terminals, but not in the cell nuclei. The distinct localization of two isoforms of CaM-KKs suggests that the complicated mechanisms for activation of CaM-KIV by CaM-KKs may be exerted in region-specific manners as well as intracellularly.