The present study was aimed at investigating the effect and the possible mechanism of idebenone on endogenous glutamate release in nerve terminals of rat cerebral cortex (synaptosomes). Idebenone inhibited the release of glutamate that was evoked by exposing synaptosomes to the K+ channel blocker 4-aminopyridine (4-AP), and this phenomenon was concentration dependent. Inhibition of glutamate release by idebenone was prevented by chelating extracellular Ca2+, or by the vesicular transporter inhibitor bafilomycin A1, but was insensitive to DL-threo-beta-benzyl-oxyaspartate, a glutamate transporter inhibitor. Idebenone decreased the depolarization-induced increase in the cytosolic free Ca2+ concentration ([Ca2+]C),whereas it did not alter the resting synaptosomal membrane potential or 4-AP-mediated depolarization. The inhibitory effect of idebenone on evoked glutamate release was prevented by blocking the Cav2.2 (N-type) and Cav2.1 (P/Q-type) channels, but not by blocking intracellular Ca2+ release or Na+/Ca2+ exchange. Furthermore, the idebenone effect on 4-AP-evoked Ca2+ influx and glutamate release was completely abolished by the protein kinase A (PKA) inhibitors, H89 and KT5720. On the basis of these results, it was concluded that idebenone inhibits glutamate release from rat cortical synaptosomes and this effect is linked to a decrease in [Ca2+]C contributed by Ca2+ entry through presynaptic voltage-dependent Ca2+ channels and to the suppression of PKA signaling cascade.