Two main forms of neurotransmitter release are known: action potential-evoked and spontaneous release. Action potential-evoked release depends on Ca²⁺ entry through voltage-gated Ca²⁺ channels whereas spontaneous release is thought to be Ca²⁺-independent. Generally, spontaneous and action potential-evoked release are believed to use the same release machinery to release neurotransmitter. This study shows, using the whole cell patch clamp technique in rat cerebellar slices, that at the interneuron to Purkinje cell synapse activation of presynaptic group II metabotropic receptors suppresses spontaneous GABA release through a mechanism independent of voltage gated Ca²⁺ channels, store-operated Ca²⁺ channels and Ca²⁺ release from intracellular Ca²⁺ stores, suggesting that the metabotropic receptors target the release machinery directly. Voltage gated Ca²⁺ channel-independent release following increased presynaptic cAMP production is similarly inhibited by these metabotropic receptors. In contrast, both voltage gated Ca²⁺ channel-dependent and presynaptic NMDA receptor-dependent GABA release were unaffected by activation of group II metabotropic glutamate receptors. Hence, the mechanisms underlying spontaneous and Ca²⁺-dependent GABA release are distinct in that only the former is blocked by group II metabotropic glutamate receptors. Thus, the same neurotransmitter, glutamate, can activate or inhibit neurotransmitter release by selecting different receptors that target different release machineries.