We have investigated the role of AMPA receptor desensitization during transmission at a calyceal synapse. Cyclothiazide blocked the rapid desensitization of AMPA receptors and markedly prolonged the decay time of the evoked excitatory postsynaptic current (PSC). This effect was greater than what would be expected from a simple prolongation of channel open time. Additionally, the drug reduced the depression of PSCs evoked at brief intervals. The effects of cyclothiazide on the PSC were reduced when the level of transmitter release was lowered. These data indicate that AMPA receptors are desensitized by neurotransmitter and that this desensitization depends on the number of quanta in the synaptic cleft. We propose that release of transmitter from many closely spaced sites prolongs the time of receptor-transmitter contact and thereby promotes desensitization. Desensitization may therefore contribute to synaptic depression and prevent the interaction of transmitter quanta within the synaptic cleft.