Ca²⁺ / calmodulin-dependent protein kinase II (CaM kinase II) consisting of α and β isoforms is highly expressed in the central nervous system and is implicated in the regulation of various Ca²⁺-dependent physiological processes. We investigated the immunohistochemical distribution of the α and β isoforms of this enzyme in the rat retina, using highly specific monoclonal antibodies which recognize each isoform. Immunoblotting revealed that not only the α but also the β isoform of CaM kinase II were expressed in the retina. The immunohistochemical study showed that highly α-immunoreactive products were localized in amacrine cells in the inner nuclear layer and displaced amacrine cells and ganglion cells in the ganglion cell layer. In addition, two well-defined bands within the inner plexiform layer were densely stained with the anti-α antibody. By contrast, immunoreactivity against the anti-β antibody was very weak in the same neuronal components of the retina. β-Immunoreactive products were homogeneously distributed throughout the inner plexiform layer and no well-defined bands were detected in this layer. Glial cells such as Müller cells were immunoreactive neither to α nor β antibody. A possible co-existence of choline acetyl transferase (ChAT) within CaM kinase II α-immunopositive neurons was examined by evaluating adjacent sections stained with anti-CaM kinase II α antibody and anti-ChAT antibody, respectively. The distribution of CaM kinase II α immunoreactivity in the rat retina was remarkably similar to that of ChAT immunoreactivity. About 32% of total ChAT-immunopositive neurons in the inner nuclear layer contained the CaM II α isoform, whereas only an occasional co-existence of both enzymes was observed in the ganglion cell layer. The present immunoblot and immunohistochemical study elucidated that not only α but also β isoforms of CaM kinase II β were expressed and this enzyme may participate in the cholinergic system in the rat retina.