Using the fluorescent membrane label FM 1-43, we have measured the release, reuptake, and repriming of synaptic vesicles in response to action potential stimulation of cultured hippocampal neurons. We find that approximately 90% of a recycling vesicle pool is released during 60 s of 10 Hz action potential firing, and that a single action potential releases approximately 0.5% of that pool. Our data also indicate that endocytic reuptake of vesicle membrane externalized by 10 Hz action potentials lags exocytosis, with a half-time on the order of 20 s, and that the minimum time for repriming of an endocytosed vesicle is on the order of 15 s. Finally, we find that once vesicles have undergone this repriming period, they become functionally mixed in the vesicle pool within a few minutes; the probability of release for recently recycled vesicles is indistinguishable from that of vesicles that have resided within the bouton for much longer periods.