The developing neuromuscular junction (NMJ) of Drosophila larvae is used as a simple model system for analysis of activity-dependent processes at glutamatergic synapses. Experience-dependent strengthening in response to enhanced locomotor activity (2h of larval crawling) has been previously reported (1). Presynaptic Drosophila NMDA receptor (DNMDAR) activity has also been reported to be enhanced in larvae following this period of activity (2), however, the molecular mechanism of DNMDAR involvement is unknown but may involve activation of the nitric oxide (NO)/cGMP pathway. We therefore characterised the expression of DNMDAR and the role of nitric oxide in synaptic plasticity processes in the Drosophila larvae. DNMDAR1 expression was assessed by Western blotting in 3rd instar larval brain and adult Drosophila head lysates. DNMDAR1 expression was also analysed by immunohistochemistry using formaldehyde-fixed larvae (1). Electrophysiological measurements of evoked excitatory junction potentials (eEJPs) were made from Drosophila muscle 6 (1,2) in the absence or presence of a soluble guanylyl cyclase inhibitor (ODQ) or in response to a NO donor (SNP). Western blot analysis of DNMDAR1 expression in larval and adult brain showed a protein doublet of approximately 130kD, which increased in intensity upon overexpression. Immunohistological analysis of DNMDAR1 showed weak labelling of neuropil structures within the ventral cord of wild type larval brains. In transgenic larvae overexpressing DNMDAR1 in motorneurons, DNMDAR1 immunoreactivity increased in the cell bodies and axons. Furthermore, a weak DNMDAR1-specific immuoreactivity at type Ib boutons of wild type NMJs, which became stronger upon overexpression of wild type DNMDAR1 in neurons, was observed. Also, DNMDAR1 was found to be localised in close proximity to presynaptic active zones. These results show that DNMDAR1 is expressed in larval brain and at NMJs. Using larvae chronically grown at 29°C to increase locomotor activity (1), application of ODQ (20μM) attenuated eEJPs from 48.6±1.4mV to 39.6±1.7mV (mean±SEM, n=6, P<0.05, paired Student's t-test), whereas SNP (10μM) enhanced eEJPs in control larvae (25°C) from 41.2±1mV to 47.9±2.1mV (n=6, P<0.05, paired Student's t-test) suggesting that the NO/cGMP pathway can regulate eEJP at the Drosophila NMJ. A link between DNMDAR1 activation and the NO/cGMP pathway remains to be established. The present data offer new insights into the mechanisms mediating experience-dependent potentiation of glutamatergic signal transmission.