The second messenger, diacylglycerol (DAG), introduces negative curvature in phospholipid monolayers and strongly induces the lamellar (L_α) to reverse hexagonal (H_II) phase transition. The chain lengths and degree of unsaturation of symmetric DAGs influence this effect. Within dioleoylphosphatidylcholine (DOPC) monolayers, the apparent spontaneous radius of curvature (R₀) of the short, saturated dicaprylglycerol (C10-DCG) itself was determined to be −13.3Å, compared with an R₀ value of −10.1Å for the long, di-monounsaturated dioleoylglycerol (C18-DOG). Such increased length and unsaturation of the DAG acyl chains produces this small change. Di-saturated phosphatidylcholines (PCs) with equal length chains (from C10-C18) with 25mol % DOG do not form the H_II phase, even under the unstressed conditions of excess water and alkane. Di-unsaturated PCs with equal chain length (from C14-C18) with 25mol % DOG do form the H_II phase. Asymmetric chained PCs (position 1 saturated with varying lengths, position 2 differentially unsaturated with varying lengths) all form the H_II phase in the presence of 25mol % DOG. As a general rule for PCs, their unsaturation is critical for the induction of the H_II phase by DOG. The degree of curvature stress induced by the second messenger DOG in membranes, and any protein that might be affected by it, would appear to depend on chain unsaturation of neighboring PCs.