Two-photon uncaging takes advantage of the inherent optical sectioning power of two-photon excitation to generate highly localized concentration increases of neurotransmitters such as glutamate. This can be used to activate isolated clusters of receptors and, thus, produce maps of receptor densities, or activate intracellular signal transduction under these receptors, in three dimensions and in complex structures such as hippocampal brain slices. Used in combination with two-photon imaging, two-photon uncaging provides a means to study the long-term structural and functional consequences of stimulation of structures such as dendritic spines. This protocol gives an overview of the procedures used for two-photon uncaging microscopy. It includes a detailed description of the development of a microscope that enables effective two-photon release of caged neurotransmitters and provides several examples of its use in cultured and acutely isolated hippocampal neurons.