The actions of diisopropylfluorophosphate (DFP), an irreversible anticholinestrase (Anti-ChE) agent, were studied on the glutamatergic neuromuscular synapse of insects. The present study reveals that DFP interacts with pre- and post-synaptic regions, also it gives alternative explanations of the symptoms of organophosphate poisoning in insects. Exposure of the metathoracic flexor tibialis muscle of Locusta migratoria to DFP (0.5 mM), at physiological solution, contained normal external calcium concentration (2 mM), increased spontaneous neurotransmitter release which was large enough to trigger action potentials (APs) and endplate potentials (EPPs). A cyclic pattern of APs and EPPs burses and silence periods were recorded. The spontaneous firing of APs and EPPs was calcium-dependent. Reduction of external calcium concentration abolished this phenomenon, suggesting a pre-synaptic site of action of DFP. A transient pre-synaptic depolarization, caused by the agent, could explain the spontaneous transmitter release and repetitive EPPs firing. The spontaneous activity, induced by DFP, was blocked by a sodium channel blocker such as tetrodotoxin. Exposure to α-bungarotoxin, α-Naja-toxin or atropine did not affect the spontaneous release of the transmitter induced by OP agent. Coupled with the pre-synaptic effect, a decrease in the peak amplitude of endplate current (EPC) and shortening of the decay time constant (τEPC) recorded after exposure to the flexor muscle to 1 mM DFP. Both the pre- and post-synaptic effects of DFP were reversible upon washing the preparation. The present findings shift the focus of the occasioners of hyperexcitation of insects treated with OPs from indirect effects of these compounds on the central nervous system (CNS) to direct effects on neuromuscular junctions and indirect effects on CNS.