Diacylglycerols which activate protein kinase C have the 1,2‐sn configuration. Short‐chain saturated fatty acids or long‐chain unsaturated fatty acids are required for supporting the potency of these lipids. Using alkyl analogs such as 1‐O‐decyl‐2‐O‐decanoylglycerol, 1‐O‐decanoyl‐2‐O‐decylglycerol, 1,2‐O‐didecyl‐glycerol, 1‐O‐hexadecyl‐2‐O‐acetylglycerol and 1‐O‐decyl‐2‐O‐acetylglycerol, we showed that the ether bond was consistently associated with a loss of activity to varying extents. The results suggest that the ester bond in the 1‐position is a major determinant of diacylglycerol‐mediated protein kinase activation.