Serotonin, a neuromodulator at the crayfish neuromuscular junction, regulates neurotransmission without changing intracellular calcium levels. However, the mechanism of this regulation remains unclear. By analysis of synaptic depression using a depletion model and measurement of vesicle recycling using the styryl dye FM1-43, we show that serotonin increases the number of vesicles available for transmitter release (total synaptic vesicle pool size). This regulation is due either to an increase in the number of vesicles at each release site or to an activation of previously nonsecreting or silent synapses. We also observed that low calcium medium rendered part of the vesicle pool unavailable for release. These results suggest a new mechanism for regulating synaptic transmission.