GABA and the GABAB receptor agonist (−)-baclofen inhibited 4-aminopyridine (4AP)- and KCl-evoked, Ca2+-dependent glutamate release from rat cerebrocortical synaptosomes. The GABAB receptor antagonist CGP 35348, prevented this inhibition of glutamate release, but phaclofen had no effect. (−)-Baclofen-mediated inhibition of glutamate release was insensitive to 2 μg/ml pertussis toxin. As determined by examining the mechanism of GABAB receptor modulation of glutamate release, (−)-baclofen caused a significant reduction in 4AP-evoked Ca2+ influx into synaptosomes. The agonist did not alter the resting synaptosomal membrane potential or 4AP-mediated depolarization; thus, the inhibition of Ca2+ influx could not be attributed to GABAB receptor activation causing a decrease in synaptosomal excitability. Ionomycin-mediated glutamate release was not affected by (−)-baclofen, indicating that GABAB receptors in this preparation are not coupled directly to the exocytotic machinery. Instead, the data invoke a direct coupling of GABAB receptors to voltage-dependent Ca2+ channels linked to glutamate release. This coupling was subject to regulation by protein kinase C (PKC), because (−)-baclofen-mediated inhibition of 4AP-evoked glutamate release was reversed when PKC was stimulated with phorbol ester. This may therefore represent a mechanism by which inhibitory and facilitatory presynaptic receptor inputs interplay to fine-tune transmitter release.