Specificity of 1,2-diacylglycerol for the activation of protein kinase C was investigated with various synthetic products. 1-Stearoyl-2-arachidonylglycerol, a major species of diacylglycerol derived from the receptor-mediated hydrolysis of inositol phospholipids, was most active, but many other diacylglycerols having naturally occurring fatty acids were almost equally active in this role. Hormone-sensitive lipase could produce potentially active diacylglycerols during lipolysis. The lack of the specificity may be reconciled with the possibility that the stearoyl-arachidonyl species is the diacylglycerol with which protein kinase C indeed comes in contact in the membrane when the receptor is stimulated, and that diacylglycerols from other sources are produced in distinct compartments and are not intercalated into the phospholipid bilayer.