Aims: SCA13 patients carrying the R420H mutation in Kv3.3 also have compromised sound localization based on inferior processing of interaural differences in time and intensity of sound. These patients show no pure-tone deficit in the audiogram and their peripheral auditory system seems unaffected. The Kv3.3 subunit of voltage-gated potassium channels is highly expressed in fast-firing neurons of the auditory pathway, including the medial nucleus of the trapezoid body (MNTB) and the lateral superior olive (LSO) and is often co-expressed with the Kv3.1 subunit. Here, we investigate how this Kv3.3 mutation affects binaural interaction.Methods: The R420H mouse model was created using CRISPR-Cas9. Brainstem audiometry and binaural interaction components (BIC) were assessed by auditory brainstem response (ABR) recordings in vivo. Single-cell responses of auditory brainstem neurons to sounds of different intensities and frequencies were compared between mature wild type, heterozygote and homozygote SCA13 mice.Results: ABR thresholds for pure-tone frequencies and clicks were similar between age-matched wild type, heterozygote and homozygote mice. However, BICs were significantly reduced in heterozygote and homozygote mice of all ages tested. Single-cell recordings showed considerably prolonged action potentials and increased synaptic delay at the level of the MNTB.Conclusions: The prolonged AP duration and increase in synaptic delay in the MNTB suggest a temporal mismatch of binaural inputs to LSO neurons. This is corroborated by the reduced binaural interaction component measured using ABRs. This non-invasive BIC test could be used as an early biomarker for auditory temporal processing.