Short-term facilitation and depression have a profound influence on transmission at many glutamatergic synapses, particularly during trains of stimuli. A major component of these processes is postsynaptic receptor desensitization. Both presynaptic and postsynaptic mechanisms can contribute to synaptic efficacy, but it is often difficult to define their respective contributions. Blockers of desensitization such as cyclothiazide (CTZ) can be used, but many of these drugs have nonspecific effects on transmitter release, complicating attempts to define synaptic effectiveness under physiological conditions. We describe and validate a new method to minimize desensitization during trains of synaptic stimuli that is based on the low-affinity competitive glutamate receptor antagonists gamma-D-glutamylglycine or kynurenic acid. A computational model of AMPA receptor kinetics shows that the mechanism can be accounted for by simple competitive antagonism of AMPA receptors, where the rapid off-rate of the antagonist permits re-equilibration between blocked and unblocked pools during the interstimulus interval. Our results at the calyx of Held show that desensitization makes little contribution to synaptic depression at frequencies below 10 Hz, but at higher frequencies it makes an important contribution, with accumulating desensitization masking short-term facilitation and causing an underestimation of quantal content. This novel method of protection from desensitization is compatible with physiological studies but cannot be used in conjunction with CTZ. Although presynaptic vesicle depletion makes the dominant contribution to short-term depression, our results show that AMPA receptor desensitization contributes to the depression at auditory synapses after hearing onset and in a frequency-dependent manner.