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Emerging evidence suggests that the phorbol ester receptor in brain may be the same as the Ca2+-phospholipid-dependent protein kinase (protein kinase C). Since protein kinase C activity is stimulated by unsaturated diacylglycerol and the phorbol esters can substitute for diacylglycerol in this stimulation, we have examined the effect of diacylglycerols on phorbol ester binding. Assays were carried out with the mouse brain cytosolic phorbol ester aporeceptor, which requires phospholipids for activity. In the presence of phosphatidylserine at 0.96 mg/ml, diolein inhibited specific binding of [3H]phorbol 12, 13-dibutyrate ([3H]PBt2) in a dose-dependent fashion to <10% of control levels. The inhibition curves fit the curve expected for a competitive inhibitor and yielded a Ki of 3.6± 0.8 μ g/ml (n = 5) [0.38% (wt/wt) the concentration of phosphatidylserine]. Scatchard analysis confirmed the competitive nature of the inhibition. At constant phospholipid concentrations, the Ki determined for diolein was independent of the diolein concentrations over the range of 1.5-80 μ g/ml, suggesting that the inhibition did not arise simply by perturbation of the phospholipid bilayers. The Ki of diolein was approximately proportional to the absolute phospholipid concentration. With phosphatidylserine at 4.8 μ g/ml, for example, the Ki was 52 ng/ml (1.1% of phosphatidylserine). In addition to diolein, the short-chain saturated diacylglycerol derivatives dicaprylin and dicaproin also inhibited [3H]PBt2 binding, whereas the long-chain saturated derivatives dipalmitin and distearin were much less active. Our results suggest (i) that diacylglycerol may act as an endogenous ligand for the phorbol ester receptor and (ii) that variation in lipid composition provides a mechanism for modulating phorbol ester receptor affinity.