The Gq subset of G protein-coupled receptors have been shown to inhibit TASK-3 channel activity. Stimulation of Gq-coupled receptors activates phospholipase C, which hydrolyzes phosphatidylinositol 4,5-bisphosphate (PIP2) generating the second messengers diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (IP3). Recent whole-cell patch-clamp experiments have suggested that depletion of PIP2 is responsible for the inhibition of TASK-3. Using the inside-out configuration of the patch-clamp technique, we examined directly the effects of PIP2 and its hydrolysis products on single TASK-3 channel activity. Phosphatidylinositol monophosphate (PIP), PIP2 and IP3 had no effect on channel activity, whereas the diacylgycerols dioctanoyl-sn-glycerol (DOG) and 1-oleoyl-2-acetyl-sn-glycerol (OAG) inhibited TASK-3. Our data suggest that increased DAG mediates the inhibitory effect of Gq-coupled receptor stimulation on TASK-3 channel function.