Modulation of synaptic efficacy may depend on the temporal correlation between pre- and postsynaptic activities. At isolated neuromuscular synapses in culture, repetitive postsynaptic application of acetylcholine pulses alone or in the presence of asynchronous presynaptic activity resulted in immediate and persistent synaptic depression, whereas synchronous pre- and postsynaptic coactivation had no effect. This synaptic depression was a result of a reduction of evoked transmitter release, but induction of the depression requires a rise in postsynaptic cytosolic calcium concentration. Thus, Hebbian modulation operates at isolated peripheral synapses in vitro, and transsynaptic retrograde interaction appears to be an underlying mechanism.