Motor nerve terminals possess multiple voltage-sensitive calcium channels operating acetylcholine (ACh) release. In this study, we investigated whether facilitation of neuromuscular transmission by adenosine generated during neuronal firing was operated by Ca2+ influx via 'prevalent' P-type or via the recruitment of 'silent' L-type channels. The release of [3H]ACh from rat phrenic nerve endings decreased upon increasing the stimulation frequency of the trains (750 pulses) from 5-Hz (83±4x103 dpm/g, n=11) to 50-Hz (30±3x103 dpm/g, n=5). The P-type Ca2+ channel blocker, w-agatoxin IVA (100 nM) reduced (40±10%, n=6) the release of [3H]ACh evoked by 50-Hz trains, while nifedipine (1 µM, an L-type blocker) was inactive. Tetanic depression was overcome (88±6x103 dpm/g, n=12) by stimulating the phrenic nerve with 50-Hz bursts (5 bursts of 150 pulses, 20 sec interburst interval). In these conditions, w-agatoxin IVA (100 nM) failed to affect transmitter release, but nifedipine (1 mM) decreased [3H]ACh release by 21±7% (n=4). Inactivation of endogenous adenosine with adenosine deaminase (ADA, 0.5 U/ml) reduced (54±8%, n=5) the release of [3H]ACh evoked with 50-Hz bursts. This effect was opposite to the excitatory actions of adenosine (0.5 mM), S-(p-nitrobenzyl)-6-thioinosine (5 µM, an adenosine uptake blocker) and CGS 21680C (3 nM, a selective A2A receptor agonist); Since the A1 receptor agonist R-N6-phenylisopropyl adenosine (R-PIA, 300 nM) failed to affect the release of [3H]ACh, the results indicate that adenosine generated during 50-Hz bursts exerts an A2A-receptor-mediated tonus. The effects of ADA (0.5 U/ml) and CGS 21680C (3 nM) were prevented by nifedipine (1 µM). Blocking tonic A2A receptor activation, with ADA (0.5 U/ml) or 3,7-dimethyl-1-propargyl xanthine (10 µM, an A2A antagonist), recovered w-agatoxin IVA (100 nM) inhibition and caused the loss of function of nifedipine (1 µM). Data indicate that, in addition to the predominant P-type Ca2+ current triggering ACh release during brief tetanic trains, motoneurons possess L-type channels that may be recruited to facilitate transmitter release during high-frequency bursts. The fine-tuning control of Ca2+ influx through P- or L-type channels is likely to be mediated by endogenous adenosine. Therefore, tonic activation of presynaptic A2A receptors operating Ca2+ influx via L-type channels may contribute to overcome tetanic depression during neuronal firing.