Calcium/calmodulin-dependent protein kinase II (CaM kinase II) is a prominent enzyme in mammalian brain capable of phosphorylating a variety of substrate proteins. In the present investigation, the subcellular and regional distribution of CaM kinase II has been studied by light and electron microscopic immunocytochemistry using an antibody that recognizes the Mr 50,000 and 60,000/58,000 subunits of the enzyme. Light microscopy demonstrates strong immunoreactivity in axons. Electron microscopy, in addition to confirming light microscopic observations, reveals moderate immunoreactivity in spines and weak immunoreactivity in nerve terminals. An accumulation of immunoreaction product is also present on postsynaptic densities. The presence of CaM kinase II in diverse structures throughout the neuron supports the view that this enzyme may be involved in mediating a variety of caldium-dependent physiological processes. CaM kinase II immunoreactivity is present in neurons throughout the brain, but a marked regional variation in the strength of the immunoreactivity exists.