The transbilayer distribution of phosphatidylethanolamine was assessed in phosphatidylcholine-phosphatidylethanolamine vesicles that contained various amounts of cytochrome b5. The small vesicles, made by sonication, and the large vesicles, made by ethanol injection, were fractionated by centrifugation before cytochrome b5 was asymmetrically incorporated into the bilayer. The mole ratio of phospholipid to protein ranged from 280 to 560 in the small vesicles and from 100 to 500 in the large vesicles. The phosphatidylethanolamine distribution, determined by chemical labeling with trinitrobenzenesulfonic acid, was assessed in vesicles the contained intact cytochrome b5 molecules and in vesicles where only the hydrophobic tail remained associated with the bilayer. At every phospholipid to protein ratio examined, the transbilayer distribution of phosphatidylethanolamine in either the small or large unilamellar vesicles was not significantly different from the distribution in control vesicles that contained no protein. Ethanol was added to some cytochrome b5-vesicle preparations (20% v/v) in an attempt to facilitate rearrangement of the phospholipids. No differences in the transbilayer distribution were observed. These results are discussed in terms of transbilayer equilibrium and the perturbation induced by the protein.