Rab3a is a small GTP-binding protein associated with presynaptic vesicles. We have measured the releasable pool in the neuromuscular junction of Rab3a(-) mice by recordings of the asynchronous release activity produced by local applications of hypertonic solutions and demonstrated that the releasable pool is significantly reduced in Rab3a(-) synapses. We found that the activity-dependent vesicle recruitment, as well as the synaptic enhancement associated with it, is disrupted in Rab3a(-) synapses. We employed Ca2+ chelators and disruption of Ca2+ sensitivity of fusion machinery by botulinum neurotoxin type-A microinjections, and demonstrated that local Ca2+ elevation may overcome the Rab3a deficiency in maintaining the releasable pool. Rab3a(-) terminals demonstrated a small but significant low-frequency depression, probably due to insufficient refilling of the releasable pool. Our results, taken together, support the hypothesis that Rab3a maintains the pool of fusion competent vesicles tightly coupled to Ca2+ channels.