Glycinergic synapses between the medial nucleus of the trapezoid body (MNTB) and the lateral superior olive (LSO) are involved in sound source localization. These synapses can reliably transmit inhibitory signals, even during minutes of high-frequency activation. Glycine transporter 2 (GlyT2) is involved in glycine reuptake from the synaptic cleft into MNTB axon terminals. Loss of GlyT2 reduces the synaptic transmission but does not lead to a complete failure (Brill et al 2021). The resilience suggests the presence of another glycine source. Here we investigated the contribution of Asc1, a high affinity glycine antiporter (Fukasawa et al 2000). We performed whole-cell patch‑clamp recordings of LSO neurons in GlyT2 knock‑out (KO) and wildtype (WT) mice while stimulating MNTB axons at 10 or 50 Hz, followed by 1 Hz recovery. Pharmacological inhibition of GlyT2 with ALX in WT resulted into a 2-fold increase of short-term depression (STD) compared to control at 50 Hz. The concurrent inhibition of GlyT2 and Asc1 with ALX+BMS led to a 4-fold stronger STD compared to control. Recovery was 1.4‑fold impaired with ALX and 2-fold with ALX+BMS. In KO, STD was reduced 3-fold with BMS compared to control. Again, recovery from STD was 4.5-fold impaired. Immunohistochemistry displayed Asc1 expression surrounding LSO somata and codistribution with the vesicular inhibitory amino acid transporter in both genotypes. Together, our results imply Asc1 expression in MNTB axon terminals. Furthermore, Asc1 blockade reduces synaptic transmission, demonstrating that Asc1, in addition to GlyT2, is a powerful glycine source at MTNB-LSO synapses.