In the cerebellum, granule cells are inhibited by Golgi cells through GABAergic synapses generating complex responses involving both phasic neurotransmitter release and the establishment of ambient gamma-aminobutyric acid (GABA) levels. Although at this synapse the mechanisms of postsynaptic integration have been clarified to a considerable extent, the mechanisms of neurotransmitter release remained largely unknown. Here we have investigated the quantal properties of release during repetitive neurotransmission, revealing that tonic GABA(B) receptor activation by ambient GABA regulates release probability. Blocking GABA(B) receptors with CGP55845 enhanced the first inhibitory postsynaptic current (IPSC) and short-term depression in a train while reducing trial-to-trial variability and failures. The changes caused by CGP55845 were similar to those caused by increasing extracellular Ca(2+) concentration, in agreement with a presynaptic GABA(B) receptor modulation of release probability. However, the slow tail following IPSC peak demonstrated a remarkable temporal summation and was not modified by CGP55845 or extracellular Ca(2+) increase. This result shows that tonic activation of presynaptic GABA(B) receptors by ambient GABA selectively regulates the onset of inhibition bearing potential consequences for the dynamic regulation of signal transmission through the mossy fiber-granule cell pathway of the cerebellum.