The medial nucleus of the trapezoid body (MNTB) serves as a relay in the binaural circuits involved in sound localization. MNTB neurons experience high-frequency sound-evoked firing up to 700Hz in vivo (Kopp-Scheinpflug et al., 2003). Such firing rates require well tuned intrinsic excitability with voltage-gated K+ channels facilitating high-frequency transmission. Kv2.2 potassium channels were recently demonstrated in the MNTB (Johnston et al., 2008) but the lack of specific blockers for the channels makes it difficult to assess their physiological role. In this study, Kv2.2 currents were investigated using whole cell patch-clamp recordings from MNTB neurons in brainstem slices prepared from CBA and Kv2.2 knockout (KO) mice. A recombinant Adenoviral (rAd) vector (Young & Neher, 2009) was developed expressing either wild type (wt) or dominant negative (dn) mouse Kv2.2 as well as EGFP. The procedures were performed in accordance with UK Animals (Scientific Procedures) Act 1986. Stereotactic surgery and microinjection were performed under anaesthesia (1.5-2.5% isoflurane) in vivo and the rAd was delivered into the MNTB of P14-15 mice. Under current-clamp, MNTB neurons exhibited elevated excitability in Kv2.2KO. Instead of a single action potential (AP) evoked by sustained depolarization as in CBA, there were trains of APs with overshooting amplitude in Kv2.2KO (CBA, 5.1±2.3mV, n=7; Kv2.2KO, 13.5±2.4mV n=12). Larger TEA-sensitive currents were observed in Kv2.2KO, suggesting Kv3 mediated compensation. Thus, rAd induced Kv2.2dn expression in CBA MNTB neurons provides a better model by specifically targeting Kv2.2 channels and avoiding general compensation. Expression of Kv2.2dn not only mimicked Kv2.2KO by converting single AP phenotype into AP trains with bigger amplitude (Kv2.2dn: 24.4±8.7, n=5) and lower firing threshold, but also broadened AP waveform (CBA: 0.30±0.03ms, n=7; Kv2.2dn: 0.48±0.04ms, n=5). Steinert et al. (see abstract at this meeting) showed that prolonged synaptic activity potentiated Kv2.2 currents via nitregic signalling in the MNTB. Rapid voltage-clamp recordings (obtained <30mins after decapitation) revealed notably greater Kv currents in CBA (36.4±5.9nA @+30mV, n=6) than those in Kv2.2 KO (15.8±1.4nA @+30mV, n=10). However, when recordings were obtained >1hr after decapitation, Kv currents were reduced by 57% in CBA (@30mV, n=20), but not in Kv2.2KO, suggesting that the magnitude of Kv2.2 currents is closely coupled with afferent activity. We conclude that Kv2.2 has a major role in regulating MNTB intrinsic excitability in vivo, and limits the excitability of the MNTB neurons during period of high activity.