The readily releasable pool of vesicles (RRP) varies in size during synaptic activity and is replenished by recruitment from the reserve pool as well as vesicle retrieval after fusion. To investigate which of these steps is rate limiting in supplying vesicles to the RRP, we measured the effects of changes in temperature in cultured hippocampal neurons, where higher average rates of release can be maintained as the temperature is increased. Using a pHluorin-based reporter of exocytosis and endocytosis (sypHy), we find that changes in temperature between 25°C and 35°C do not significantly alter the rate of recruitment from the reserve pool. In contrast, the time constant of endocytosis fell from ∼17 s at 25°C to ∼10 s at 35°C (Q10= 1.7), while the time constant of vesicle reacidification fell from ∼5.5 s to ∼1 s (Q10= 5.5). A kinetic model of the vesicle cycle constructed using measured parameters was found to describe variations in vesicle release rate observed during long trains of spikes as well as recovery from synaptic depression after bursts of activity. These results indicate that endocytosis operating with time constants of 10–15 s is the rate-limiting process determining replenishment of the RRP during long-term activity. A fast mode of vesicle retrieval could not be detected at any temperature, nor was it necessary to invoke such a mechanism to account for use-dependent changes in synaptic release probability.