Neuronal microtubules recently emerged as temporal and spatial regulators of dendritic spines, the major sites of excitatory synaptic input. By imaging microtubules in cultured mature primary hippocampal neurons using fluorescently tagged tubulin and microtubule plus-end binding (EB) protein EB3, dynamic microtubules were found to regularly depart from the dendritic shaft and enter dendritic spines. Evidence indicates that microtubule invasions into spines regulate spine actin dynamics and induce transient morphological changes, such as the formation of spine head protrusion and spine growth. Because alterations in spine morphology play an important role in synaptic plasticity and have been linked to learning and memory formation, it is possible that dynamic microtubules are engaged in adaptive processes in the adult brain. This chapter provides detailed methods for live imaging of dynamic microtubules in mature hippocampal neurons in culture. We describe protocols for culturing and transfecting mature hippocampal neurons and visualizing microtubules and microtubule plus-EB proteins by total internal reflection fluorescence microscopy and spinning disk confocal microscopy.