AbstractCa2+/calmodulin‐dependent protein kinase I (CaM kinase I) was originally identified in rat brain based on its ability to phosphorylate site 1 of synapsin I. Recently a cDNA for the rat brain enzyme has been cloned and the primary structure elucidated [Picciotto et al. (1993), J. Biol. Chem., 268:26512–26521]. The rat cDNA encoded a protein of 374 amino acids with a calculated Mr of 41,636. Antibodies have now been raised against the recombinant kinase expressed in E. coli as a glutathione‐S‐transferase fusion protein. Immunoblot analysis of rat cortex lysates revealed two major immunoreactive bands of ∼Mr 38,000 and 42,000. Minor immunoreactive species of slightly lower Mr were also detected. Two distinct CaM kinase I activities were partially purified from rat brain and shown to correspond to the two major immunoreactive species. A variety of immunoreactive species of Mr 35–43,000 were detected in “brain” tissue from cow, zebra finch, goldfish, Xenopus, lamprey, and Drosophila. In rat brain, immunocytochemistry revealed strong staining in cortex, hippocampus, amygdala, hypothalamus, brain stem, and choroid plexus. The labelling was mainly observed in neuropil but clusters of intensely labelled neuronal cell bodies were also detected all along the neuraxis. Neuronal nuclei and glial cells did not appear to be stained. Subcellular fractionation studies confirmed the cytosolic localization of the kinase in the brain. In various rat non‐neuronal tissues and in a number of cell lines, immunoreactive species of ∼Mr 38,000 and ∼42,000 were detected at lower levels than that detected in brain. The Mr 38,000 and 42,000 species were also found in different ratios and at different levels in the non‐neuronal tissues. These results support a role for CaM kinase I in the regulation of multiple neuronal processes. Furthermore, the widespread cell and tissue distribution suggests that CaM kinase I may function as a ubiquitous multi‐functional protein kinase. Finally, the multiple immunoreactive species may represent isoforms of CaM kinase I. © 1995 Wiley‐Liss, Inc.