Local anesthetics have been widely used for regional anesthesia and the treatment of cardiac arrhythmias. Recent studies have also demonstrated that low-dose systemic local anesthetic infusion has neuroprotective properties. Considering the fact that excessive glutamate release can cause neuronal excitotoxicity, we investigated whether local anesthetics might influence glutamate release from rat cerebral cortex nerve terminals (synaptosomes). Results showed that two commonly used local anesthetics, lidocaine and bupivacaine, exhibited a dose-dependent inhibition of 4-AP-evoked release of glutamate. The effects of lidocaine or bupivacaine on the evoked glutamate release were prevented by the chelation of extracellular Ca2+ ions and the vesicular transporter inhibitor bafilomycin A1. However, the glutamate transporter inhibitor dl-threo-beta-benzyl-oxyaspartate did not have any effect on the action of lidocaine or bupivacaine. Both lidocaine and bupivacaine reduced the depolarization-induced increase in [Ca2+]C but did not alter 4-AP-mediated depolarization. Furthermore, the inhibitory effect of lidocaine or bupivacaine on evoked glutamate release was prevented by blocking the Cav2.2 (N-type) and Cav2.1 (P/Q-type) channels, but it was not affected by blocking of the ryanodine receptors or the mitochondrial Na+/Ca2+ exchange. Inhibition of protein kinase C (PKC) and protein kinase A (PKA) also prevented the action of lidocaine or bupivacaine. These results show that local anesthetics inhibit glutamate release from rat cortical nerve terminals. This effect is linked to a decrease in [Ca2+]C caused by Ca2+ entry through presynaptic voltage-dependent Ca2+ channels and the suppression of the PKA and PKC signaling cascades. Synapse 67:568–579, 2013. © 2013 Wiley Periodicals, Inc.