Recently, it has been claimed that vesicles recycling spontaneously and during activity belong to different pools. Here we measured simultaneously, by using spectrally separable styryl dyes, the release kinetics of vesicles recycled spontaneously or upon stimulation, as well as the influence of the vATPase blocker folimycin on the frequency of miniature postsynaptic currents in rat hippocampal neurons. Our results provide evidence for the identity of vesicle pools recycling at rest and during stimulation.