At each stage of infection, the major coat protein of coliphage M13 binds to the E. coli cytoplasmic membrane with its antigenic site exposed to the cell exterior [Wickner, W. (1975) Proc. Nat. Acad. Sci. USA 72, 4749-4753]. This antigenic site is now shown to be at the amino-terminus of the protein. The amino-terminus of M13 coat protein is also found exclusively on the outside of dilauroyl or dimyristoyl lecithin vesicles, formed with coat protein by the cholate dilution technique [Racker, E., et al. (1975) FEBS Lett. 57, 14-18] near the lipid phase transition temperature. The basic carboxyterminus of the coat protein is exclusively on the inside of these vesicles. Vesicles of M13 coat protein and dimyristoyl lecithin when formed below the lipid phase transition temperature have both ends of the coat protein exposed to the vesicle exterior. The asymmetry of a membrane protein can, therefore, be established in the absence of other proteins and of lipid asymmetry; it depends on the physical state of the lipid phase. The factors which cause asymmetry in this model system may affect the distribution of proteins in biological membranes.