Glycogen synthase kinase-3beta (GSK3beta) is highly abundant in the brain. Various biochemical analyses have indicated that GSK3beta is localized to different intracellular compartments within brain cells. However, ultrastructural visualization of this kinase in various brain regions and in different brain cell types has not been reported. The goal of the present study was to examine GSK3beta distribution and subcellular localization in the brain using immunohistochemistry combined with light and electron microscopy. Initial examination by light microscopy revealed that GSK3beta is expressed in brain neurons and their dendrites throughout all the rostrocaudal extent of the adult mouse brain, and abundant GSK3beta staining was found in the cortex, hippocampus, basal ganglia, the cerebellum, and some brainstem nuclei. Examination by transmission electron microscopy revealed highly specific subcellular localization of GSK3beta in neurons and astrocytes. At the subcellular level, GSK3beta was present in the rough endoplasmic reticulum, free ribosomes, and mitochondria of neurons and astrocytes. In addition GSK3beta was also present in dendrites and dendritic spines, with some postsynaptic densities clearly labeled for GSK3beta. Phosphorylation at serine-9 of GSK3beta (pSer9GSK3beta) reduces kinase activity. pSer9GSK3beta labeling was present in all brain regions, but the pattern of staining was clearly different, with an abundance of labeling in microglia cells in all regions analyzed and much less neuronal staining in the subcortical regions. At the subcellular level pSer9GSK3beta labeling was located in the endoplasmic reticulum, free ribosomes and in some of the nuclei. Overall, in normal brains constitutively active GSK3beta is predominantly present in neurons while pSer9GSK3beta is more evident in resting microglia cells. This visual assessment of GSK3beta localization within the subcellular structures of various brain cells may help in understanding the diverse role of GSK3beta signaling in the brain.