The effect of alpha-tocopherol, the major vitamin E component, on the release of endogenous glutamate has been investigated using rat cerebrocortical nerve terminals. Results showed that alpha-tocopherol facilitated the Ca2+-dependent but not the Ca2+-independent glutamate release evoked by 4-aminopyridine (4AP). This release facilitation was insensitive to glutamate transporter inhibitor L-trans-PDC or DL-TBOA, and blocked by the exocytotic neurotransmitter release inhibitor tetanus neurotoxin, indicating that alpha-tocopherol affects specifically the physiological exocytotic vesicular release without affecting the non-vesicular release. Facilitation of glutamate exocytosis by alpha-tocopherol was not due to its increasing synaptosomal excitability, because alpha-tocopherol did not alter the 4AP-evoked depolarization of the synaptosomal plasma membrane potential. Rather, examination of the effect of alpha-tocopherol on cytoplasmic free Ca2+ concentration revealed that the facilitation of glutamate release could be attributed to an increase in voltage-dependent Ca2+ influx. Consistent with this, the alpha-tocopherol-mediated facilitation of glutamate release was significantly reduced in synaptosomes pretreated with omega-CgTX MVIIC, a wide spectrum blocker of N- and P/Q-type Ca2+ channels. In addition, alpha-tocopherol modulation of glutamate release appeared to involve a protein kinase C (PKC) signalling cascade, insofar as pretreatment of synaptosomes with the PKC inhibitor GF109203X effectively suppressed the facilitatory effect of alpha-tocopherol on 4AP- or ionomycin-evoked glutamate release. Furthermore, alpha-tocopherol increased the phosphorylation of MARCKS, the major presynapic substrate for PKC, and this effect was also significantly attenuated by PKC inhibition. Together, these results suggest that alpha-tocopherol exerts an increase in PKC activation, which subsequently enhances voltage-dependent Ca2+ influx and vesicular release machinery to cause an increase in evoked glutamate release from rat cerebrocortical glutamatergic terminals. This finding might provide important information regarding to the action of vitamin E in the central nervous system.