Neurons undergo complex morphological changes during differentiation and in cases of plasticity. A major determinant of cell morphology is the actin cytoskeleton, which in neurons is comprised of two actin isoforms, non-muscle γ- and β-actin. To better understand their respective roles during differentiation and plasticity, their cellular and subcellular localization was examined in developing and adult cerebellar cortex. It was observed that γ-actin is expressed at a constant level throughout development, while the level of β-actin expression rapidly decreases with age. At the light microscopic level, γ-actin staining is ubiquitous and the only developmental change observed is a relative reduction of its concentration in cell bodies and white matter. In contrast, β-actin staining almost completely disappears from the cytoplasm of cell bodies, primary dendrites and axons. In young cerebellar cultures, γ-actin is found in the cell body, neurites and growth cones, while β-actin is mainly found in growth cones, as previously reported in other primary neuronal culture systems [Kaech et al. (1997), J. Neuroscience, 17, 9565–9572; Bassell et al. (1998), J. Neuroscience, 18, 251–265]. Electron microscopy of post-embedding immunogold-labelled tissue confirms the widespread distribution of γ-actin, and also reveals an increased concentration of γ-actin in dendritic spines in the adult. During development, β-actin accumulation is observed in actively growing structures, e.g. growth cones, filopodia, cell bodies and axonal tracts. In the adult cerebellar cortex, β-actin is preferentially found in dendritic spines, structures which are known to retain their capacity for morphological modifications in the adult brain. This differential subcellular localization and developmental regulation of the two actin isoforms point to their different roles in neurons.