Nitric oxide (NO) is an activity-dependent modulator of synaptic transmission in the brain; it regulates intrinsic excitability and potentially, mediates homeostasis across neuronal populations, through a process of volume transmission. The short half-life and low effective concentrations of NO makes it difficult to study its physiological roles and underlying mechanisms. Neurons in the medial nucleus of the trapezoid body (MNTB) generate NO in response to synaptic stimulation. The surrounding nuclei receive strong inhibitory projections from the MNTB, which suggests that there is also the potential for nitrergic signalling. Here we ask whether these MNTB target nuclei are modulated via NO volume transmission. CBA/CaJ mice (P14-21) were killed by decapitation in accordance with the UK Animals (Scientific Procedures) Act 1986. Whole-cell patch recording from brainstem slices were used to study the three MNTB target nuclei: the medial and lateral superior olive (MSO & LSO) and the superior paraolivary nucleus (SPN). The hyperpolarization-activated non-specific cation conductance (IH) was studied in current-clamp and voltage-clamp mode under control conditions and following activation of nitrergic signalling. Hyperpolarizing current injection was used to mimic an inhibitory input and showed the characteristic slow ‘sag’ of IH in neurons of the MSO, LSO and SPN. Hyperpolarizing voltage steps in voltage-clamp mode showed inward IH currents activating negative to -50 mV. IH currents were smaller in the MNTB compared to the target nuclei (MNTB: 4.9 ±0.3nS; SPN: 21.3 ±1.3nS; MSO: 18.5 ±1.3nS; LSO: 40.3 ±5.7). Activation time-constants at -130mV were slower in the MNTB and equally fast in the target nuclei (MNTB: 47.5 ±2.8ms; SPN: 28.9 ±1.8ms; MSO: 22.8 ±1.5ms; LSO: 25.1 ±2.9ms). Half-activation voltages were most negative in the MNTB and most positive in the SPN (MNTB: -94.9 ±1.8mV; SPN: -87.8 ±0.8mV; MSO: -90.3 ±1.4mV; LSO: -88.4 ±1.2mV). Modulation by NO was tested by perfusion of two different NO donors (100μM SNP or DEANONOate). NO caused a significant hyperpolarizing shift of 10mV and 15mV in IH half-activation voltage in SPN and MSO neurons, respectively. This was accompanied by a slowing of the IH activation time-constants (MSO: 29.9 ±2.3; SPN: 51.9 ±7.8ms). NO did not affect IH peak current amplitudes in any of the tested nuclei. The NO-induced shift of half-activation voltages was unaffected by raising intracellular cGMP but was prevented by incubation with antagonists of soluble guanylyl cyclase (1μM ODQ). Immunohistochemistry shows high expression of HCN1 subunits in MSO and SPN neurons where the greatest modulation in half-activation was also observed, consistent with a conclusion that NO modulates HCN1 but not HCN2 subunits.