The structural dynamics of synapses probably has a crucial role in the development and plasticity of the nervous system. In the mammalian brain, the vast majority of excitatory axo-dendritic synapses occur on dendritic specializations called ‘spines’. However, little is known about their long-term changes in the intact developing or adult animal. To address this question we developed a transcranial two-photon imaging technique to follow identified spines of layer-5 pyramidal neurons in the primary visual cortex of living transgenic mice expressing yellow fluorescent protein. Here we show that filopodia-like dendritic protrusions, extending and retracting over hours, are abundant in young animals but virtually absent from the adult. In young mice, within the ‘critical period’ for visual cortex development, ∼73% of spines remain stable over a one-month interval; most changes are associated with spine elimination. In contrast, in adult mice, the overwhelming majority of spines (∼96%) remain stable over the same interval with a half-life greater than 13 months. These results indicate that spines, initially plastic during development, become remarkably stable in the adult, providing a potential structural basis for long-term information storage.