Some ion channels are regulated by inositol phospholipids and by the products of cleavage by phospholipase C (PLC). KCNQ channels (Kv7) require membrane phosphatidylinositol 4,5-bisphosphate (PIP(2)) and are turned off when muscarinic receptors stimulate cleavage of PIP(2) by PLC. We test whether diacylglycerols are also important in the regulation of KCNQ2/KCNQ3 channels using electrophysiology and fluorescent translocation probes as indicators for PIP(2) and diacylglycerol in tsA cells. The cells are transfected with M(1) muscarinic receptors, channel subunits, and translocation probes. Although they cause translocation of a fluorescent probe with a diacylglycerol-binding C1 domain, exogenously applied diacylglycerol (oleoyl-acetyl-glycerol and dioctanoyl glycerol) and phorbol ester do not mimic or occlude the suppression of KCNQ current by muscarinic agonist. Blocking the metabolism of endogenous diacylglycerol by inhibiting diacylglycerol kinase with R59022 or R59949 slows the decay of diacylglycerol twofold but does not mimic or occlude muscarinic regulation and recovery of current. Blocking diacylglycerol lipase with RHC-80267 also does not occlude muscarinic modulation of current. We conclude that the diacylglycerol produced during activation of PLC, any activation of protein kinase C that it may stimulate, and downstream products of its metabolism are not essential players in the acute muscarinic modulation of KCNQ channels.