The neuronal form of nitric oxide synthase (nNOS) is widely expressed in the brain and via coupling to NMDA receptor-mediated calcium influx, it is linked to synaptic plasticity (Bon & Garthwaite, 2003). However, its physiological activation and the mechanisms by which nitric oxide (NO) acts to affect neuronal excitability have proved elusive. Here, we exploit the unique input-specificity of the calyx of Held synapse to characterise NO release at this model glutamatergic synapse in the auditory pathway in acute brain slices. CBA/CaJ mice (P10-P15) were killed by decapitation in accordance with the UK Animals (Scientific Procedures) Act 1986. Brainstem slices containing the superior olivary complex were prepared as previously described (Wong et al., 2003) and imaging experiments were performed at 36°C on principal neurons of the medial nucleus of the trapezoid body (MNTB). Synaptic activity was achieved by midline stimulation using a bipolar electrode. Immunohistochemistry showed that nNOS is present in the cytoplasm of principle neurons in the MNTB. Slices and/or single cells were loaded with DAR-4M AM (10μM) and FURA 2 AM (10μM) and excited at 560 and 380nm to monitor NO and [Ca²⁺]_i, respectively. We show that synaptic activity at the calyx of Held causes a NMDA receptor-dependent release of NO in postsynaptic neurons. The fluorescence ratio of two repeated stimuli (F2/F1) was 1.5±0.1 (n=13 cells) for control and was suppressed by AP-5 (50μM), DNQX (10μM) and the two NOS antagonists N-PLA (10μM) and 1400W (10μM) (0.45±0.08*; 0.88±0.06*; 0.33±0.07*; 0.77±0.09* and 0.46±0.1*, respectively, n=3-12 cells, p<0.05). Synaptic activity also increased cGMP levels (basal: from 4.87±0.84 pmol mg^-1 protein to: 21.86±3.67* pmol mg^-1 protein, n=7-9 slices, p<0.05) which required action potential propagation and nNOS activity. Release of NO from active neurons also raised NO-dependent DAR-4M fluorescence in adjacent non-active neurons indicating the volume diffusion of NO. This diffusion between neurons was suppressed by the NO scavenger PTIO (100μM). This data supports the hypothesis that NO serves as a volume transmitter at the calyx of Held/MNTB, integrating spontaneous and evoked neuronal firing to provide an index of global activity, thereby regulating excitability and/or synaptic strength across a population of neurons and providing a significant homeostatic control of physiological functions. Results are reported as mean±SEM. The comparison of different groups was carried out using two-tailed Student’s t-test. One way ANOVA was applied for testing statistical significance between more then 2 data sets. Differences were considered statistically significant at p<0.05.