Previous in vivo studies of crustacean neuromuscular synapses have shown that a chronic increase in the impulse activity of a previously ‘inactive’ motoneuron produces a reduction in initial transmitter release and greater resistance to synaptic fatigue. To explore the mechanisms of this synaptic change, we have developed an in vitro procedure for examining this activity-dependent reduction in initial transmitter release. We report that depolarization selectively applied to the proximal region of the neuron (cell body or axon) of a phasic motoneuron produces a reduction in initial transmitter release from the motor terminals. This synaptic change is observed 4–5 h after the beginning of depolarization. Proximal depolarization decreases initial transmitter release without reducing the capacity of the terminals to release transmitter during repetitive stimulation. Application of a calcium channel blocker during conditioning prevents the reduction in initial transmitter release. These results demonstrate that prolonged calcium influx produce a long-term reduction in initial transmitter release, and that calcium influx in distant regions of the motoneuron can influence transmitter release from motor terminals. The relationship of these findings to previously reported activity-dependent synaptic changes is discussed.