We measured neurotransmitter release and motor nerve terminal currents in mouse phrenic nerve-diaphragm and triangularis sterni preparations, to evaluate the role of Ca2+-channel subtypes in regulating transmitter release. Saturated concentrations of either ωagatoxin IVA [ω-Aga-IVA (0.3 μM), a blocker of P-type Ca2+channels] or ω-conotoxin MVIIC [ω-CTx-MVIIC (2 μM), a P-and Q-type Ca2+-channel blocker], inhibited nerve-evoked muscle contractions and the amplitude of endplate potentials respectively. In contrast, combined treatment with nifedipine (50 μM, a blocker of L-type Ca2+ channels) plus ω-conotoxin GVIA [ω-CTx-GVIA (2 μM), a blocker of N-type Ca2+ channels] did not elicit inhibitory effects on nerve-evoked muscle contractions, endplate potentials or nerve terminal waveforms. Because of the non-linear relationship between endplate potentials and Ca2+ signals, a small decrease in presynaptic Ca2+ entry can significantly reduce the amplitude of the endplate potential. Thus, we applied 3, 4-diaminopyridine (3, 4-DAP, a k+-channel blocker) or high Ca2+(10 mM) to accelerate and amplify the endplate potentials and Ca2+ currents. The endplate potentials amplified by 3, 4-DAP or by high Ca2+ correspondingly proved to be quite resistant to both ω-Aga-IVA and ω-CTx-MVIIC; ωAga-IVA exerted only a partial inhibitory effect on endplate potentials, and the ω-Aga-IVA-resistant component was further inhibited by ω-CTx-MVIIC. The component that was resistant to the two toxins could be completely blocked by the non-selective Ca2+ channel blocker Cd2+ (300 μM). A combination of the two toxins had no significant effects on either spontaneous transmitter release or postsynaptic resting membrane potentials of the diaphragm preparation and the Na+ and K+ waveforms of the triangularis sterni preparations. This finding suggests a preferential inhibitory effect at a presynaptic site. Measuring the Ca2+ currents in the triangularis sterni also revealed partial inhibition by ω-CTx-MVIIC with further incomplete inhibition by ω-Aga-IVA. Cd2+ (300 μM) abolished the toxin-resistant component of the Ca2+ current. In contrast, a combination of nifedipine (50 μM) with ω-CTx-GVIA (2 μM) was without inhibitory effect. We conclude that multiple types of Ca2+channels, i.e. ω-Aga-IVA-sensitive, ω-CTx-MVIIC-sensitive and toxin-resistant Ca2+ channels, coexist in mouse motor nerve terminals.