Neurotransmitter release is controlled by calcium (Ca2+) entry into motor nerve terminals (MNTs) through voltage-dependent calcium channels (VDCCs). Upon Ca2+ influx, acetylcholine is released starting downstream processes causing muscle contraction. Co-release of acetylcholine and glutamate from MNTs has been reported. Ca2+ influx through N-methyl-D-aspartate (NMDA) receptors on postsynaptic site is evident. In this study we aimed to observe nerve-evoked and directly-elicited twitch responses in the presence of VDCCs inhibitors without blocking NMDA receptors. We elicited contractions with nerve-evoked (0.1 Hz; 0.3 ms) and direct (0.1 Hz; 3 ms) electrical stimulations at rat hemidiaphragm preparations by using some VDCC toxins and drugs. We evaluated their effects at 15 minutes of application. P/Q-type VDCC blocker omega-conotoxin MVIIC (25 nM) suppressed the nerve-evoked and direct stimulation contractions relative to their initial value by 50 and 40%, respectively. N-type VDCC inhibitor omega-conotoxin GVIA (170 nM) had no effect on both stimulation contractions. P-type VDCC blocker omega-agatoxin IVA (10 nM) completely blocked the nerve-evoked stimulation contractions, whereas the direct stimulation contractions maintained the same values as baseline. L-type VDCC blockers verapamil (75 mu M) and diltiazem (75 mu M) depressed both nerve-evoked and direct stimulation contractions. Ethanol (650 mM) blocked nearly 100% of the contractions by nerve-evoked stimulation, meanwhile 45.5% of direct stimulation. Our finding may suggest the Ca2+ influx localization matters for decoding the neural information for the contractile force generation.