Voltage-gated potassium (Kv) channels are widely expressed in the central nervous system and serve to regulate neuronal excitability. High voltage-activated K⁺ channels are responsible for shaping action potential (AP) waveforms, thereby enhancing high frequency transmission (Rudy & McBain, 2001). Here, we exploit the calyx of Held synapse to characterize the effects of nitric oxide (NO) on K⁺ channels at this glutamatergic synapse in vitro. CBA/CaJ mice (P10-P15) were killed by decapitation in accordance with the UK Animals (Scientific Procedures) Act 1986. Brainstem slices were prepared as previously described (Wong et al., 2003) and patch clamp experiments were performed at 36°C on principal neurons of the medial nucleus of the trapezoid body (MNTB). Synaptic responses were achieved by midline stimulation using a bipolar electrode, inducing activity-dependent release of NO which suppressed high voltage-activated K⁺ currents over a time period of 10-20 min (control: 13.1±1.0 nA vs synaptic stimulation: 7.0±1.3* nA, n=5, p<0.05, paired t-test). Comparable suppression was achieved by perfusion of two different NO donors (control: 16.9±0.3 nA, 100μM SNP: 6.9±1.0* nA, 100μM DEANONOate: 5.4±1.5* nA, n=58-5, p<0.05). This reduction in current was prevented by incubation with antagonists of soluble guanylate cyclase and PKG (1μM ODQ: control: 14.3±2.2 nA vs SNP: 13.5±2.4 nA and 1μM KT5823: control: 19.7±0.3 nA vs SNP: 19.0±0.9 nA). Inhibition of postsynaptic Kv3 currents by TEA (3 mM), NO donors (SNP, DEANONOate, both 100μM) or synaptic activity each independently increased AP half-widths which was also modeled by a 60% reduction in the Kv3-mediated current with a single compartment model of a MNTB neuron (constructed using NEURON simulation software). Similar changes in AP waveforms were observed from in vivo recordings following sound exposure. NO signaling modulated the ability of neurons to faithfully follow high frequency transmission causing failures at frequencies higher then 50Hz and converting transmission from a phasic to an onset pattern. In trains of APs with Poisson-distributed inter-spike-intervals, the mean MNTB firing rate dropped from 82.1±5.5Hz to 38.5±7.9* Hz (n=4, p<0.05, paired t-test). In addition to the NO actions in synaptically targeted neurons, NO suppressed Kv3 currents by diffusion to adjacent inactive neurons (control: 12.9±1.1 nA vs synaptic stimulation: 8.8±0.9* nA, n=5, p<0.05, paired t-test) thereby modulating excitability in a diffusion-limited volume (see Abstract by Steinert et al. at this meeting). We conclude that NO is an endogenous modulator of postsynaptic excitability and information transmission in the auditory brainstem. Results are reported as mean±SEM. Comparisons were carried out using two-tailed Student’s t-test. Differences were considered statistically significant at p<0.05.