Cerebellar granule cells are inhibited phasically by GABA released synaptically from Golgi cells, but are inhibited more powerfully by tonic activity of high affinity alpha 6 subunit-containing GABAA receptors. During development the tonic activity is generated by the accumulation of GABA released by action potentials, but in the adult the tonic activity is independent of action potentials. Here we show that in adult rats the tonic activation of GABAA receptors is produced by non-vesicular transmitter release and is reduced by the activity of GAT-1 and GAT-3 GABA transporters, demonstrating that alterations of GABA uptake will modulate information flow through granule cells. Acetylcholine (ACh) evokes a large Ca2+-dependent but action potential-independent release of GABA, which activates alpha 6 subunit-containing GABAA receptors. These data show that three separate modes of transmitter release can activate GABAA receptors in adult cerebellar granule cells: action potential-evoked exocytotic GABA release, non-vesicular release, and ACh-evoked Ca2+-dependent release independent of action potentials. The relative magnitudes of the inhibitory charge transfers generated by action potential-evoked release (during high frequency stimulation of the mossy fibres), tonic inhibition and superfused ACh are 1:3:12, indicating that tonic and ACh-mediated inhibition may play a major role in regulating granule cell firing.