The mechanism for assembly of membrane lipid bilayers in vivo is generally considered to occur on existing membranes which act as templates for the assembly process. Not presently understood is the source of the membrane template. A thermodynamic theory describing a spontaneous process for assembly of unilamellar vesicles with properties of a critical state (1,2) has been tested in this study. The spontaneous formation of large unilamellar vesicles (LUV’s) from phospholipid components of cell membranes at a critical temperature T* has now been documented by cryo-electron microscope studies using aqueous dispersions of dimyrystoylphosphatidylcholine (T* = 29°C), and the total lipid extracts of E. coli membranes cultured at 32°C (T* = 32°C). The lipids were incubated at temperatures below, at and above the critical temperatures for LUV assembly. in conformity with thermodynamic theory, and other physical measurements that indicate unique bilayer properties exist at T* (3-5), LUV’s are seen to form only at T*.