•  Spontaneous activity contributes to low synaptic depression of the calyx of Held synapse in vivo. •  Application of a reversible blocker in combination with juxtacellular recordings allows a relatively good estimate of local drug concentrations reached during microiontophoresis. •  Activation of the GABAB receptor on the young‐adult calyx of Held can reduce short‐term depression, both in vivo and in slices. •  The ambient concentration of GABA in the auditory brainstem is low. Abstract  The calyx of Held synapse of the medial nucleus of the trapezoid body is a giant axosomatic synapse in the auditory brainstem, which acts as a relay synapse showing little dependence of its synaptic strength on firing frequency. The main mechanism that is responsible for its resistance to synaptic depression is its large number of release sites with low release probability. Here, we investigated the contribution of presynaptic GABAB receptors and spontaneous activity to release probability both in vivo and in vitro in young‐adult mice. Maximal activation of presynaptic GABAB receptors by baclofen reduced synaptic output by about 45% in whole‐cell voltage clamp slice recordings, which was accompanied by a reduction in short‐term depression. A similar reduction in transmission was observed when baclofen was applied in vivo by microiontophoresis during juxtacellular recordings using piggyback electrodes. No significant change in synaptic transmission was observed during application of the GABAB receptor antagonist CGP54626 both during in vivo and slice recordings, suggesting a low ambient GABA concentration. Interestingly, we observed that synapses with a high spontaneous frequency showed almost no synaptic depression during auditory stimulation, whereas synapses with a low spontaneous frequency did depress during noise bursts. Our data thus suggest that spontaneous firing can tonically reduce release probability in vivo. In addition, our data show that the ambient GABA concentration in the auditory brainstem is too low to activate the GABAB receptor at the calyx of Held significantly, but that activation of GABAB receptors can reduce sound‐evoked synaptic depression.