The biopharmaceutical characteristics of double liposomes (DLs) containing insulin were examined, and the usefulness of DLs in combination with aprotinin is discussed. Encapsulation of insulin was influenced by lipid composition, and the highest efficiency was observed with positively charged liposomes. Insulin encapsulated in liposomes, especially in DLs, was protected from enzymatic proteolysis. A portion of insulin molecules was adsorbed on the surface of the membrane when liposomes were prepared using a lipid with a positive charge and was degraded by enzymes. Remarkable hypoglycemic effects were observed after intragastric administration of DLs containing insulin at a dose of 20 IU/kg to normal male Wistar rats. The highest mean relative efficacy to administration was obtained with insulin-loading DLs containing aprotinin as a protease inhibitor. These results suggest that DLs are applicable as an oral dosage form for peptide drugs such as insulin etc., especially in combination with protease inhibitors.