To test the hypothesis that activation of protein kinase C (PKC) is related to the interface between coexisting diacylglycerol-(DAG-) enriched and DAG-poor phases, the thermotropic phase behavior of the ternary mixtures dimyristoylphosphatidylcholine (DMPC)/dimyristoylphosphatidylserine (DMPS)/dioleoylglycerol (DO), DMPC/DMPS/1-palmitoyl-2-oleoylglycerol (PO), and DMPC/DMPS/dimyristoylglycerol (DM) was analyzed and compared with the ability of the lipid mixtures to support PKC activity. Differential scanning calorimetry (DSC) was used to monitor the gel-to-liquid crystalline phase transition as a function of the mole fraction of DO (χ(DO)), PO (χ(PO)), or DM (χ(DM)) in DMPC/DMPS (1:1) multilamellar vesicles (MLVs) and of χ(DO) in large unilamellar vesicles (LUVs). The addition of DAG at low mole fractions gave rise to the appearance of two or more overlapping transitions. The phase boundaries of the ternary mixtures deduced from the partial phase diagrams were χ(DO) = ~0.10 and ~0.3 for DMPC/DMPS/DO, χ(PO) = ~0.05 and ~0.4 for DMPC/DMPS/PO, and χ(DM) = ~0.025 and ~0.5-0.6 for DMPC/DMPS/DM. Above these mole fractions of DAG, the transitions again became very sharp. The ability of the lipid mixtures to support activity of PKC α and PKC η was examined below and above the gel-to-liquid crystalline phase transition. In the gel phase, PKC activity went through a maximum as a function of increasing mole fraction of each DAG and was restricted to lipid compositions in which coexisting phases were observed. Maximal activity decreased with increasing saturation of the DAG. In the fluid state, maximal PKC activity was shifted to higher DO mole fractions and the peak was much broader. Collectively, these data support a role for both the presence and nature of interface between compositionally distinct domains in activation of PKC.