Recently, we reported the mRNA localization of Ca(2+)/calmodulin-dependent protein kinase I beta 2 isoform (CaMKIbeta2) in the mouse nervous system. In the present study, polyclonal antibody against CaMKIbeta2 was generated and used to investigate the distribution of the enzyme within the central nervous system of the rat. Interestingly some differences were observed between the enzyme localization and previous mRNA detection [J. Neurochem. 268 (1999) 26512]. The strongest expression of the enzyme was found in pontine nuclei. Immunopositive fibers could be traced through the middle cerebellar peduncle until they reached the cerebellum. Quite strong staining could also be observed in almost all of the neurons in the neocortex, hippocampus, amygdala, hypothalamus, brainstem and cerebellum, including the nuclei of the cranial nerves and Purkinje cell layer of the cerebellar cortex which was not clearly detected in the previous in situ hybridization study. In the spinal cord, CaMKIbeta2 could be detected in the gray matter with stronger expression in the dorsal horn. CaMKIbeta2 showed very strong nuclear localization but was also present in the cytoplasm of some neurons. Such localization suggests that CaMKIbeta2 may be involved in many neuronal functions in the central nervous system, including the possibility of important roles in nuclear signal transduction.