Polyclonal antibody against Ca2+/calmodulin-dependent protein kinase V (CaM kinase V) was prepared from guinea pigs immunized with synthetic polypeptide, based on the partial amino acid sequence of the rat brain enzyme. Immunoblot analysis of purified CaM kinase V revealed two immunoreactive bands with a molecular mass of 41 kDa and minor 40 kDa, respectively. Tissue distribution of CaM kinase V and immunohistochemical localization in rat brain were also investigated. Immunoblotting revealed the presence of immunoreactive proteins with the same molecular mass of 40 and 41 kDa, in the cerebrum, cerebellum, brain stem, pituitary gland, lung, adrenal gland, spleen, liver, colon, heart, stomach, ovary, spinal cord, and thymus. Immunohistochemistry revealed strong staining in the neuronal somata and weak staining in the nuclei. Densely stained regions included the cerebral cortex, the hippocampal formation, the caudatoputamen, the globus pallidus, the hypothalamus, the substantia nigra, the medial geniculate body, the olfactory bulb, the cerebellar cortex and the choroid plexus. CaM kinase V revealed different substrate specificity compared with CaM kinase II. These results lead to the notion that CaM kinase V may exist in 40- and 41-kDa isoforms, is widely distributed in various tissues, and may play an important role in the control of a wide variety of calcium regulated processes in the respective tissues.