Cerebellar granule (CG) neurons express a G protein-gated K+ current (GIRK) that is involved in the neurotransmitter regulation of the excitatory input to the Purkinje fibres of the cerebellum. Here, we characterized the single-channel behaviour of GIRK in CG neurons, and examined the effects of several known modulators of GIRK and their putative physiological roles. Whole-cell GIRKs were activated by baclofen, a GABAB receptor agonist. In cell-attached patches, baclofen activated GIRK with a single-channel conductance of 34 pS and a mean open time of 0.5 ms. In inside-out patches, application of GTPgammaS to the cytoplasmic side activated GIRK with similar kinetic properties. Addition of 2 mM ATP resulted in a marked increase in GIRK activity and induced longer-lived openings with a mean open time of 2.3 ms (ATP-dependent gating). Brain cytosolic fraction or free fatty acids inhibited this effect of ATP, and this was reversed by addition of purified recombinant brain fatty acid binding protein. Applying phosphatidylinositol 4,5-bisphosphate (PIP2) to inside-out patches in place of ATP also increased GIRK activity; however, only an increase in the frequency of opening was observed. The stimulatory effect of PIP2 on GIRK activity was not inhibited by the cytosolic fraction. Following maximal activation by PIP2, ATP caused an additional 2.2-fold increase in GIRK activity. These results show that GIRKs in CG neurons are regulated by positive and negative modulators that affect frequency as well as open time duration. The net effect is that the ligand-activated GIRK is in the 'low activity' state associated with short-lived openings, mainly due to strong action of the cytosolic inhibitor of ATP-dependent gating. Our results also show that intracellular ATP modulates GIRK via pathways different from that of PIP2 in CG neurons.