Neuromuscular junction endplate potentials (EPPs) decrease quickly and to a large extent during continuous stimulation. The present study examined the hypothesis that this may occur partially due to a negative feedback loop, activated by presynaptic α7 neuronal nicotinic ACh-receptors (α7-NAChR). We stimulated a nerve in a neuromuscular preparation of isolated mouse hemidiaphragm, cut to remove the contractile response, to produce long trains of evoked EPPs at 50 Hz for prolonged periods of time (40-60 seconds). All values are presented as means with standard error of mean; statistical testing was performed using Student’s paired t-test. Substantial rundown of EPP amplitude and quantal content (measured directly as the ratio between evoked EPP and miniature EPP amplitudes) was found in the course of nerve stimulation: after 40 seconds of stimulation the quantal content of EEP was decreased to 55.2±3.1% from the initial level (n=8). This decrease was significantly attenuated by the pre-application of either α-cobratoxin (5 nM) or methyllicaconitine (100 nM) (74.2±7.2% and 76.2±7.9% respectively; n=8, p<0.05 in both cases), known as selective α7-NAChR antagonists. Neither antagonist changed the amplitude of miniature EPPs when applied on their own, leading us to believe that they possess no postsynaptic action. Externally applied choline, a selective α7-NAChR agonist, decreased the quantal content of single EPPs (by 20%) and EPPs in short (1-2 sec) 50 Hz bursts of EPPs (71.6±6.1% of initial EPP after a period of 1 second stimulation vs. 88.6±2.1% in control; n=11, p<0.05). This choline-induced rundown of EPPs in a short train was similar to that caused by prolonged stimulation without exogenous choline. The depressory effect of choline was also clearly removed by concomitant application of α-cobratoxin (94.7±3.2% from initial EPP after 1 second; n=5), bringing the EPP amplitude and quantal content back to control levels. The data obtained allows to suggest that endogenous choline, produced in the synaptic cleft by acetylcholine esterase from ACh during periods of prolonged synaptic activity, binds to a previously uncharacterized pool of pre- or perisynaptic α7-NAChR, which can down-regulate the mediator secretion in the neuromuscular junction, via a negative feedback loop. This feedback doesn’t normally result in transmission failure due to a very high safety ratio of the neuromuscular synapse and is probably aimed to conserve mediator reserves over the time of prolonged rhythmic synaptic activity. Nevertheless, this negative feedback could also result in synaptic failure under certain pathological conditions that are shown to negatively affect neuromuscular transmission.