This paper is the second in a series of studies on the light and electron microscopic distribution of filamentous actin (F-actin) in the rat central nervous system (CNS) using phalloidin tagged with the fluorophore eosin followed by fluorescence photooxidation. A previous report described the selective localization of high concentrations of F-actin in subpopulations of dendritic spines in hippocampus, cerebellum and neostriatum. Dendritic spines were the most intensely stained structures in the CNS, but several other structures were notable for their consistent staining for F-actin. Although the majority of cell bodies, axons and large dendrites were unlabeled, mossy fibers and Schaffer collaterals in the hippocampal formation, basket cell axons in the cerebellar pinceau, and granule cell dendrites in the glomeruli of the cerebellar cortex routinely showed strong F-actin labeling. Staining was observed in all three glial cell types. Labeling was consistently observed in the astrocytic processes surrounding the Purkinje cell soma and primary dendrite. Intense but sporadic staining was observed in the perinodal glia of the Node of Ranvier. A few examples of labeled oligodendrocyte processes were also seen in the neostriatum. Labeling was observed in microglia in every brain region examined, although the labeling was present in the lumen of the endoplasmic reticulum and the nuclear membrane, leading to questions about its specificity. Perycites apposed to the blood vessels also showed very consistent labeling. Our results suggest that selected structures in the adult CNS in addition to dendritic spines are enriched in F-actin.