Genetically encoded fluorescent probes have become indispensable tools in the biological sciences. Studies of synaptic vesicle recycling have been facilitated by a group of GFP-derived probes called pHluorins. These probes exploit changes in pH that accompany exocytosis and recapture of synaptic vesicles. Here we describe how these synaptic tracers can be used in rodent hippocampal neurons to monitor the synaptic vesicle cycle in real time and to obtain mechanistic insights about it. Synapses can be observed in living samples using a wide-field fluorescence microscope and a cooled charge-coupled device camera. A simple specimen chamber allows electrical stimulation of synapses to evoke exocytosis in a precisely controlled manner. We present protocols to measure various parameters of the synaptic vesicle cycle. This technique can be easily adapted to study different classes of synapses from wild-type and mutant mice. Once cultured neurons expressing synaptopHluorin are available, the whole procedure should take about 2 h.