Phospholipid membranes are useful in the field of biocatalysis because a supported phospholipid membrane can create a biomimetic platform where biocatalytic processes can readily occur. In this work, supported bilayer formation from sonicated phospholipid vesicles containing 1,2-dielaidoyl-sn-glycero-3-phosphocholine and 1,2-dimyristoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] was studied using a quartz crystal microbalance with dissipation monitoring and an atomic force microscope. The molar percentages of DEPC and DMPG were varied to determine the effect of overall lipid composition on supported bilayer formation. This work also explored the effect that calcium ion concentration had on supported bilayer formation. Results show that vesicles with up to 50 mol% dimyristoylphosphoglycerol can form a supported bilayer without the presence of calcium ions; however, supported bilayer formation in calcium buffer was inhibited as the anionic (negatively charged) lipid concentration increased. Data suggest that supported phospholipid bilayer formation in the absence of Ca2+ from vesicles containing negatively charged lipids is specific to phosphatidylglycerol. Published by Elsevier B.V.