The mechanism of action of the A 2A adenosine receptor agonist 2- p-(2-carboxyethyl) phenethylamino-5′-N-ethylcarboxamidoadenosine hydrochloride (CGS-21680) in the facilitation of spontaneous (isotonic and hypertonic condition) and K +-evoked acetylcholine (ACh) release was investigated in the mouse diaphragm muscles. At isotonic condition, the CGS-21680-induced excitatory effect on miniature end-plate potential (MEPP) frequency was not modified in the presence of CdCl 2 and in a medium free of Ca 2+ (0Ca 2+-EGTA), but it was abolished after buffering the rise of intracellular Ca 2+ with 1,2-bis-(2-aminophenoxy)-ethane- N,N,N′,N′-tetraacetic acid tetra(acetoxy-methyl) (BAPTA-AM) and when the Ca 2+-ATPase inhibitor thapsigargin was used to deplete intracellular Ca 2+ stores. CGS-21680 did not have a direct effect on the Ca 2+-independent neurotransmitter-releasing machinery, since the modulatory effect on the hypertonic response was also occluded by BAPTA-AM and thapsigargin. CGS-21680 facilitation on K +-evoked ACh release was not altered by the P/Q-type voltage-dependent calcium channel (VDCC) blocker ω-Agatoxin IVA, but it was completely prevented by both, the L-type VDCC blocker nitrendipine (which is known to immobilize their gating charges), or thapsigargin, suggesting that the effects of CGS-21680 on L-type VDCC and thapsigargin-sensitive internal stores are associated. We found that the VDCC pore blocker Cd 2+ (2 mM Ca 2+ or 0Ca 2+-EGTA) failed to affect the CGS-21680 effect in high K + whereas nitrendipine in 0Ca 2+-EGTA+Cd 2+ occluded its action. The blockade of Ca 2+ release from endoplasmic reticulum with ryanodine antagonized the facilitating effect of CGS-21680 in control and high K + concentration. It is concluded that, at the mouse neuromuscular junction, activation of A 2A receptors facilitates spontaneous and K +-evoked ACh release by an external Ca 2+-independent mechanism but that involves mobilization of Ca 2+ from internal stores: during spontaneous ACh release stimulating directly the ryanodine-sensitive stores and, at high K +, probably modulating the L-type VDCCs which may cause the opening of the ryanodine receptors that would be directly coupled to the channels. In both cases, Ca 2+ released from the endoplasmic reticulum would be capable of activating the exocytotic machinery, thus producing facilitation of ACh release.