The glycoprotein of vesicular stomatitis (VS) virus was selectively liberated from the virion membrane by the dialyzable nonionic detergent, beta-D-octylglucoside. The isolated viral glycoprotein could be rendered virtually free of phospholipid and detergent, under which conditions it formed tail-to-tail glycoprotein micelles in the form of rosettes. When mixtures of viral glycoprotein and egg lecithin were dialyzed free of octylglucoside, glycoprotein vesicles formed spontaneously with spikes protruding in the same external orientation as the VS virion membrane. The glycoprotein vesicles exhibited increased and uniform buoyant density, indicating relative homogeneity in the proportion of glycoprotein and phosphatidylcholine in each glycoprotein liposome. Evidence for similar insertion and orientation of VS viral glycoprotein in both phosphatidylcholine vesicles and virion membrane was substantiated by the finding that proteolytic digestion with thermolysin gave rise to hydrophobic glycoprotein tail fragments in vesicle or virion membranes that migrated identically in polyacrylamide gels.