Neurons transmit information at chemical synapses by the release of neurotransmitter contained within small vesicles. The maintenance of synaptic transmission requires that these vesicles be recycled after fusion with the cell surface, but these endocytic mechanisms have been. It is established that synaptic vesicles can collapse on fusion and the machinery for retrieving this collapsed membrane by clathrin-mediated endocytosis (CME) is enriched at hippocampal boutons (Takei et al., 1996). What is less clear is the speed at which CME operates at small central synapses and its importance compared to other mechanisms of vesicle retrieval. In fact, it has been suggested that the large majority of vesicles released by physiological stimulation are recycled by a second, faster mechanism called “kiss-and-run,” which operates in 1 s or less (Aravanis et al. 2003). A key feature of the kiss-and-run model is that the vesicle is retrieved at the site of fusion before it has collapsed into the surface membrane (Gandhi & Stevens, 2003). What is the relative importance of these different endocytic mechanisms during normal synaptic activity? To address this question, we designed sypHy, a new and improved optical reporter of exocytosis and endocytosis made by fusing pH-sensitive GFP (Miesenbock et al., 1998) to the synaptic vesicle protein synaptophysin. The fluorescence of sypHy increases when vesicles fuse, and then declines when they are re-acidified after endocytosis. Vesicle reacidification occurred with a time-constant of 4 s, allowing us to estimate the rate of endocytosis from the decline in the sypHy signal. The average release probability reported by sypHy was slightly larger than the value of 0.35 measured electrophysiologically, indicating that pHluorin reliably records all fusion events and can therefore provide an unbiased view of the kinetics of endocytosis. We measured synaptic vesicle retrieval after single action potentials (APs), when fast kiss-and-run has been reported to be the predominant mode of fusion, but found only one mode of endocytosis, which occurred with a time constant of 15 s at room temperature. The speed of endocytosis was the same at synapses of high and low release probability and constant for a range of stimulus strengths up to 40 APs at 20 Hz. Monitoring the connection of a fused vesicle with the surface of the cell by altering the external pH provided an alternative method of estimating the rate of endocytosis which yielded a time-constant of 12 s. Two different methods of inhibiting CME blocked vesicle retrieval after weak stimuli: overexpression of a dominant-negative construct (AP180-C) and knockdown of clathrin heavy chain using RNAi. These results provide clear evidence against the idea that fast, clathrin-independent mechanisms of vesicle retrieval play a significant role at hippocampal synapses. We therefore conclude that CME is the physiological mechanism of vesicle retrieval (Granseth et al. 2006).