The effect of (E)-2-methyl-6-styryl-pyridine (SIB1893), a selective metabotropic glutamate (subtype 5) receptor (mGlu(5)R) antagonist, on glutamate release from isolated nerve terminals (synaptosomes) was examined. SIB1893 caused a potent inhibition of the Ca(2+)-dependent release of glutamate evoked by 4-aminopyridine (4AP). That the implied mGlu(5)R-mediated modulation was contingent on diacylglycerol stimulation of protein kinase C (PKC) was indicated by PKC activator phorbol dibutyrate and PKC inhibitor Ro 32-0432 (bisindolylmaleimide XI), respectively, superceding or suppressing the inhibitory effect of SIB1893. The inhibitory action of SIB1893 was not due to it decreasing synaptosomal excitability or directly interfering with the release process at some point subsequent to Ca(2+) influx, because SIB1893 did not alter the 4AP-evoked depolarization of the synaptosomal plasma membrane potential or Ca(2+) ionophore ionomycin-induced glutamate release. Rather, examination of the effect of SIB1893 on cytosolic [Ca(2+)] revealed that the diminution of glutamate release could be attributed to a reduction in voltage-dependent Ca(2+) influx. Consistent with this, the SIB1893-mediated inhibition of glutamate release was completely prevented in synaptosomes pretreated with a combination of the N- and P/Q-type Ca(2+) channel blockers, omega-conotoxin GVIA, and omega-agatoxin IVA. Together, these results suggest that noncompetitive antagonism of mGlu(5)Rs using SIB1893 effects a decrease in PKC activation, which subsequently attenuates the Ca(2+) entry through voltage-dependent N- and P/Q-type Ca(2+) channels to cause a decrease in evoked glutamate release. These actions of SIB1893 and related agents may contribute to their neuroprotective effects in excitotoxic injury.