Spontaneous secretion of the neurotransmitter acetylcholine in mammalian neuromuscular synapsis depends on the Ca²⁺ content of nerve terminals. The Ca²⁺ electrochemical gradient favors the entry of this cation. We investigated the possible involvement of three voltage-dependent Ca²⁺ channels (VDCC) (L-, N-, and P/Q-types) on spontaneous transmitter release at the rat neuromuscular junction. Miniature end-plate potential (MEPP) frequency was clearly reduced by 5 µM nifedipine, a blocker of the L-type VDCC, and to a lesser extent by the N-type VDCC blocker, -conotoxin GVIA (-CgTx, 5 µM). On the other hand, nifedipine and -CgTx had no effect on K⁺-induced transmitter secretion. -Agatoxin IVA (100 nM), a P/Q-type VDCC blocker, prevents acetylcholine release induced by K⁺ depolarization but failed to affect MEPP frequency in basal conditions. These results suggest that in the mammalian neuromuscular junction Ca²⁺ enters nerve terminals through at least three different channels, two of them (L- and N-types) mainly related to spontaneous acetylcholine release and the other (P/Q-type) mostly involved in depolarization-induced neurotransmitter release. Ca²⁺-binding molecule-related spontaneous release apparently binds Ca²⁺ very rapidly and would probably be located very close to Ca²⁺ channels, since the fast Ca²⁺ chelator (BAPTA-AM) significantly reduced MEPP frequency, whereas EGTA-AM, exhibiting slower kinetics, had a lower effect. The increase in MEPP frequency induced by exposing the preparation to hypertonic solutions was affected by neither external Ca²⁺ concentration nor L-, N-, and P/Q-type VDCC blockers, indicating that extracellular Ca²⁺ is not necessary to produce hyperosmotic neurosecretion. On the other hand, MEPP frequency was diminished by BAPTA-AM and EGTA-AM to the same extent, supporting the view that hypertonic response is promoted by "bulk" intracellular Ca²⁺ concentration increases.