Dendritic spines are the postsynaptic sites of most excitatory synapses in the mammalian brain. With the advent of two-photon microscopy and transgenic mice expressing fluorescent proteins, dendritic spines can now be imaged in the living cerebral cortex over time scales ranging from seconds to years. Recent studies with this in vivo imaging approach have begun to provide important insights into the development and plasticity of dendritic spines in the intact brain. Here, we review these studies and discuss technical requirements for image acquisition. We envision that intravital two-photon imaging at the level of individual synapses will greatly expand our current understandings of how neuronal networks are assembled and modified throughout life.