Regulation of neurotransmitter release is thought to involve modulation of the release probability by protein phosphorylation. Activation of the cAMP-protein kinase A (PKA) pathway has been shown to facilitate synaptic transmission in mammalian neuromuscular synapses, although the relevant phosphorylation targets are mostly unknown. We found that the inhibitor of the phosphodiesterase aminophylline (1 mM AMIN), the membrane-permeable analog of cAMP, 8-Br-cAMP (5 mM) and, the direct adenylate cyclase activator, forskolin (20 microM), induced an increase of miniature end-plate potentials (MEPPs) frequency in rat neuromuscular junctions. We investigated the possible involvement of the voltage-dependent calcium channels (VDCC), since these proteins are known to be phosphorylated by PKA. But this possibility was ruled out, since the increase in MEPPs frequency was not attenuated by the VDCC blocker Cd2+ (100 microM) and it was observed when AMIN was studied on hyperosmotic response, which is independent of [Ca2+]o and of Ca2+ influx through the VDCC. The lack of action of AMIN on MEPPs frequency when [Ca2+]i was diminished by exposing the preparations to zero Ca2+-EGTA solution (isotonic condition) or when nerve terminals were loaded with a permeant Ca2+ chelator (BAPTA-AM) (hypertonic condition), indicate that cAMP-mediated presynaptic facilitation is a function of nerve terminal Ca2+ concentration. We also found that AMIN exerted a comparable increase in MEPPs frequency in control and high K+ (10 and 15 mM), suggesting a single mechanism of action for spontaneous and K+-induced secretion.