Diacylglycerol modulates cell functions primarily through activation of protein kinase C (PKC). In a previous study, however, we found that a diacylglycerol analogue, 1-oleoyl-2-acetylglycerol (OAG), accelerated desensitization of neuronal nicotinic acetylcholine receptors (nAchRs) independently of PKC activation in PC12 cells. In the present study, we investigated whether other analogues and endogenous diacylglycerol exert similar effects on neuronal nAchRs and characterized the modulation by diacylglycerol. We measured the nicotine-induced whole-cell current in the absence and presence of diacylglycerol analogues in PC12 cells. We also investigated the effects of a blockade of metabolic pathways of diacylglycerol by inhibiting diacylglycerol lipase and kinase. We found that all four diacylglycerol analogues studied promoted desensitization and depressed the nondesensitized component of the nicotine-induced current. These effects seemed independent of PKC activation because they were not antagonized by the PKC inhibitors staurosporine or bisindolylmaleimide I; one analogue that lacks the PKC-stimulating action was also effective. The effects of diacylglycerol analogues were not antagonized by high doses of nicotine and were independent of the membrane potential. Similar modulatory effects were observed by treatment with RHC80267, a blocker of diacylglycerol lipase, and R59949, an inhibitor of diacylglycerol kinase, in the presence of staurosporine. These results suggest that diacylglycerol, both exogenously applied and endogenously produced, modulates neuronal nAchRs independently of PKC activation in PC12 cells; further, these effects seemed consistent with a noncompetitive and voltage-independent block. They raised the possibility that PKC-independent inhibition of neuronal nAchRs by diacylglycerol may be a novel modulatory process.