The classical ‘delayed rectifier’ potassium current is mediated by Kv3 channels which open at voltages during action potential (AP)-mediated depolarisation and thus provide the repolarization drive to support high-frequency firing in neurons. Multiple Kv3 genes (KCNC1-4) are co-expressed in several parts of the brain, either as hetero- or homo-tetramers (1-3) but subunit specific roles are largely unknown. Neurons of the medial nucleoid trapezoid body (MNTB) and the lateral superior olive (LSO) in the superior olivary complex (SOC) of the auditory brainstem compute sound source localisation through integration of binaural stimuli. Both MNTB and LSO neurons express Kv3.1 and Kv3.3 subunits, but lack Kv3.2 and Kv3.4 subunits (4). We have used their neuronal network to investigate the necessity or redundancy of Kv3.1 and Kv3.3 subunits in generating functional Kv3 channels and during repolarization of the postsynaptic AP. The study was conducted on wildtype (WT), Kv3.1 knockout (KO), and Kv3.3 KO mice in a CBA/Ca background strain in accordance with the Animals (Scientific Procedures) Act UK 1986 and as revised by European Directive 2010/63/EU. Expression of Kv3 channels in the MNTB and LSO principal neurons was investigated by qRT-PCR, western blot and immunohistochemistry. For ex vivo whole cell patch-clamp electrophysiology, transverse sections of SOC were obtained from brainstem and used for recording currents and voltages in the neurons under different pharmacological and biophysical conditions. Intracellular Ca2+ transients were studied by fura-2 ratiometric fluorescence measurement. Statistical significance was determined using Student’s t-test and one-/two-way ANOVA and expressed as mean ± SD. The results obtained reveal that MNTB neurons effectively employed either Kv3.1 or Kv3.3 subunits in Kv3 channels, with similar whole-cell current amplitude, however, the fastest APs were achieved when both subunits were expressed together (WT: 0.29 ± 0.07ms, n=14; Kv3.1 KO: 0.41 ± 0.11ms, n=11, p=0.0207; Kv3.3 KO: 0.48 ± 0.06ms, n=11, p<0.0001). In the LSO, Kv3.3 subunits were essential; Kv3.1 mRNA was present, but somatic Kv3 channels must contain at least one Kv3.3 subunit, since Kv3.3KO mice had little or no TEA-sensitive Kv3 current (Ctrl: 6.0 ± 2.5nA, n=11; TEA: 5.0 ± 1.8nA, n=7), and AP halfwidths (0.69 ± 0.15ms, n=7, p<0.0001) were increased compared to WT (0.28 ± 0.04ms, n=10). Measurement of whole-cell voltage-gated Ba2+ currents showed no change in the MNTB or LSO, but the longer AP duration in the KOs increased activity-dependent [Ca2+]i in an activity-dependent manner. We conclude that Kv3 channels make major contributions to AP repolarization in both the MNTB and LSO. Kv3.1 and Kv3.3 subunits each contribute to Kv3 currents in the MNTB, while Kv3.3 is crucially dominant for Kv3 channels in the LSO, both affecting Ca2+ influx in these neurons.