The trapping efficiency of globular proteins in 4 different types of phosphatidylcholine vesicles was systematically studied. Vesicles were generated in a mixture of 125I-labeled proteins of various MW. The trapped proteins were separated from untrapped proteins by gel filtration and ultrafiltration and subsequently analyzed by gel electrophoresis and autoradiography. Entrapment of proteins was seen by their resistance to trypsin digestion. The relative amount of each entrapped protein species was compared to that of the original protein solution. In multilamellar vesicles and large unilamellar vesicles, proteins of MW up to 97,000 had the same trapping efficiency as sucrose. In small unilamellar vesicles generated by sonication or ethanol injection, the relative trapping efficiency of protein decreased progressively as the MW of the protein became greater. The trapping efficiency of .alpha.-amylase (Mr [molecular weight relative to 1/2 the weight of 1 atom of 12C] 97,000) was 1/2 of that for sucrose. The apparent decrease in trapping efficiency with the protein''s MW in small unilamellar vesicles can be accounted for by the combination of the bound water layer at the vesicle''s internal surface and the steric hindrance when protein is captured during vesicle formation.