Whether a quantal packet of transmitter saturates postsynaptic receptors is a fundamental question in central synaptic transmission. However, this question remains open with regard to saturation at mature synapses. The calyx of Held, a giant glutamatergic synapse in the auditory brainstem, becomes functionally mature during the fourth postnatal week in rats. During postnatal development, the mean amplitude of miniature (i.e., quantal) EPSCs (mEPSCs) becomes significantly larger. Experiments using the rapidly dissociating glutamate receptor antagonist kynurenate suggested that vesicular glutamate content increases with development. To test whether AMPA receptors are saturated by a packet of transmitter, we infused a high concentration of l-glutamate into mature calyceal terminals. This caused a marked increase in the mean amplitude of mEPSCs. We conclude that a single packet of transmitter glutamate does not saturate postsynaptic AMPA receptors even at the mature calyx of Held synapse with increased vesicular transmitter content.