Transmitter is loaded into vesicles using secondary active transport, that is, the vacuolar H+-ATPase pumps protons into the vesicle and protons are then exchanged for neurotransmitter. Surprisingly, mutations in the V-ATPase did not decrease the amount of neurotransmitter. Rather, the frequency of the vesicle fusions was reduced. Our results are consistent with an acidification checkpoint whereby only fully acidified synaptic vesicles are competent to fuse. The uniform neurotransmitter content may be controlled not by a direct mechanism that monitors neurotransmitter content in a vesicle, but instead by an indirect mechanism affected by the acidification state of the vesicle.