Summary The structural and chemical specificity of diacylglycerols, lipid components of the quaternary complex for protein kinase C activation, have been evaluated. The ether-linked analogs of the diacyl lipids, either dialkyl or alkyl acyl, were not effective activators of protein kinase C and thus had little influence on reducing the Ca ++ requirement of the enzyme. Ester-linked compounds such as 1-palmitoyl- sn-2-butyrylglycerol were as effective as dioleoylglycerol in stimulating protein phosphorylation. Increasing the carbon number at the sn-2 position from two to four resulted in enhanced enzymatic activity, suggesting that the chain length at the secondary hydroxyl is also of importance. These data clearly establish the necessity of the sn-1 carbonyl group of ester-linked glycerolipids in the protein kinase C activation complex.