Functional imaging microscopy based on voltage-sensitive dyes (VSDs) has proven effective for revealing spatiotemporal patterns of activity in vivo and in vitro. Microscopy based on two-photon excitation (TPE) of fluorescent VSDs offers the possibility of three-dimensional recording of membrane potential changes on subcellular length scales hundreds of microns below the brain’s surface. Here we describe progress in monitoring membrane voltage using TPE of VSD fluorescence, and detail an application of this emerging technology in which action potentials (APs) were recorded in single trials from individual mammalian nerve terminals in situ. Prospects for, and limitations of this method are reviewed.