Nerve stimulation increases the concentration of ATP in the synaptic cleft, which can act as a neurotransmitter or as a presynaptic neuromodulator. Using the luciferin-luciferase assay, we observed that the extracellular concentration of ATP increased by 11-26 nM over a basal concentration of 6 nM, in a frequency dependent manner (1-5 Hz), in the adult rat phrenic nerve-hemidiaphragm preparation. This ATP release depends on nerve activity since it was abolished by tetrodotoxin (1 microM) and is strictly dependent on the presence of extracellular calcium. However, more than half of this nerve-evoked release of ATP is derived from activated muscle fibres since the selective post-synaptic nicotinic receptor antagonist, alpha-bungarotoxin (1 microM), inhibited by over 60% the evoked release of ATP. The presently observed post-synaptic release of ATP together with the previously reported lack of post-synaptic effects of ATP and to the ability of ATP to act as a presynaptic modulator open the possibility that ATP may behave as a retrograde messenger at this neuromuscular junction.