The TASK subfamily of two pore domain potassium channels (K2P) gives rise to leak potassium currents, which contribute to the resting membrane potential of many neurons and regulate their excitability. K2P channels are highly regulated by phosphorylation and by G protein-mediated pathways. In this study, we show that protein kinase C (PKC) inhibits recombinant TASK3 channels. Inhibition by PKC is blocked by the PKC inhibitors bisindolylmaleimide 1 hydrochloride (BIM) and 12-(2-cyanoethyl)-6,7,12,13-tetrahydro-13-methyl-5-oxo-5H-indolo(2,3-a)pyrrolo(3,4-c)-carbazole (Gö6976). Gene-silencing experiments with a validated small interfering RNA sequence against PKCalpha ablates the effect of PKC. PKC acts directly on hTASK3 channels to phosphorylate an identified amino acid in the C terminus region (Thr341), thereby reducing channel current. PKC also inhibits mTASK3 channels despite their having a quite different C-terminal structure to hTASK3 channels. Activation of M(3) muscarinic receptors inhibits both hTASK3 channels expressed in tsA-201 cells and standing outward potassium current (IK(SO)) in mouse cerebellar granule neurons through the activation of the G protein Galpha(q), because both effects are abolished by the selective Galpha(q) antagonist YM-254890 (J Biol Chem 279:47438-47445, 2004). This inhibition is not directly transduced through activation of PKC because inhibition persists in mutated PKC-insensitive hTASK3 channels. Instead, inhibition seems to occur through a direct action of Galpha(q) on the channel. Nevertheless, preactivation of PKC blocks muscarinic inhibition of both TASK3 channels and IK(SO). Our results suggest that activation of PKC (via phospholipase C) has a role in opposing inhibition after M(3) receptor activation rather than transducing it and may act as a negative regulator of G protein modulation to prevent prolonged current inhibition.