Abnormal nitric oxide (NO) signalling is implicated in several neurodegenerative diseases (Steinert et al., 2010) but its exact contribution to neuronal death remains elusive due to great complexity of downstream nitrergic targets. Several NO-mediated mechanisms are associated with neuronal degeneration, including mitochondrial and synaptic dysfunction or activation of cell death pathways. Elevated NO can lead to formation of cytotoxic peroxynitrite which in turn can modulate a wide range of protein functions via nitration of tyrosine residues (3-Nitrotyrosination [3-NT]). Toxic NO signalling can further alter protein functioning in a process known as S-nitrosylation. To date, little is known as to what extent NO-mediated post-translational modifications contribute to or exacerbate neuronal dysfunction (Nakamura et al., 2013). We use glutamatergic synapses as a model system to identify novel nitrergic signalling pathways to correlate protein modifications with functional changes.Here we investigate the effects of NO on synaptic function which may involve S-nitrosylation or 3-NT signalling. The Drosophila neuromuscular junction was used as a model to characterise NO-mediated effects at the synapse employing immunohistochemical and two-electrode-voltage-clamp (TEVC) analyses. Electrophysiological recordings were carried out in HL-3 solution in 1.5mM Ca2+ using sharp electrodes (20-30 MΩ). Data denote mean±SEM (n-number) with *p<0.05 indicating statistical significance using unpaired Student’s t-test. Confocal analysis showed elevated levels of S-nitroso-cysteine and 3-Nitrotyrosine at synapses following NO-donor exposure (GSNO, SNAP, 500μM each for 1h, 3 NMJs each). TEVC data showed little NO effects on miniature excitatory junctional current (mEJCs) amplitudes or frequencies but induced a reduction in mEJC decay kinetics (τ; Ctrl: 6.1±0.1ms (49) vs NO: 5.5±0.2ms* (24)). Furthermore, evoked EJC (eEJC) amplitudes (Ctrl: 101±5nA (18) vs NO: 48±6nA* (5)) and quantal content (QC; Ctrl: 119±6 (18) vs NO: 75±7* (5)) were strongly reduced following NO exposure for >35min indicative for a reduction in release probability (pvr). An increased paired-pulse ratio at various interspike-intervals under NO conditions confirmed a reduction in pvr (20ms: 1.1±0.0 vs 1.3±0.1*; 40ms: 1.0±0.0 vs 1.3±0.1* (n=7-11)). Cumulative postsynaptic current analysis (500ms 50Hz train) further showed a reduced number of release-ready vesicles following NO exposure (382±45 (7) vs 154±44* (5)) which was also confirmed by fluctuation analysis (Silver, 2003).Together, our data suggest that NO can induce 3-NT or S-nitrosylation of proteins involved in synaptic signalling possibly leading to protein modifications as detected by changes in synaptic physiology. This data extends our understanding of NO signalling, potentially leading to the identification of putative targets for therapeutic intervention(s) in disease.