Electrical activity during early development affects the development and maintenance of synapses (Spitzer [2006]: Nature 4447:707-712), but the intercellular signals regulating maintenance of synapses are not well identified. At the neuromuscular junction, adenosine 5-triphosphate (ATP) is coreleased with acetylcholine at activated nerve terminals to modulate synaptic function. Here we use cocultured mouse motor neurons and muscle cells in a three-compartment cell culture chamber to test whether endogenously released ATP plays a role in activity-dependent maintenance of neuromuscular synapses. The results suggest that ATP release at the synapse counters the negative effect of electrical activity, thus stabilizing activated synapses. Confirming our previous work (Li et al. [2001]: Nat Neurosci 4:871-872), we found that in doubly innervated muscles, electrical stimulation induced heterosynaptic downregulation of the nonstimulated convergent input to the muscle fiber with no or little change of the stimulated inputs. However, in preparations that were stimulated in the presence of apyrase, an enzyme that degrades extracellular ATP, synapse downregulation of stimulated inputs was substantial and significant, and end plate potentials were reduced. Apyrase treatment for 20 h in the absence of stimulation did result in moderate diminution, but this was prevented by blocking spontaneous neural activity with tetrodotoxin. The P2 receptor blocker, suramin, also induced activity-dependent synapse diminution. The decrease in synaptic efficacy produced by prolonged stimulation in the presence of apyrase persisted for greater than 20 h, consistent with a developmental time-course and distinct from the rapid neuromodulatory actions of ATP that have been demonstrated by others. We conclude that extracellular ATP promotes stabilization of the neuromuscular junction and may play a role in activity-dependent synaptic modification during development.