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□ The interactions between insulin and various lipids were studied by monolayer penetration experiments at constant surface area. The increase in surface pressure, ΔΠ, of a lipid film depended upon the particular lipid used and the concentration of insulin in the subphase. For all lipids studied, ΔΠ was dependent on the initial surface pressure of the lipid film. Evidence of the interaction between insulin and the lipids was found in the ability of insulin to penetrate lipid films with initial pressures >16 dynes/cm, the maximum surface pressure obtained by insulin alone. For phospholipids, both the nonpolar and polar regions influenced the degree of interaction with insulin. Saturated chain lecithins exhibited less penetration than phospholipids with unsaturated hydrocarbon chains. The net charge of the lipid was not found to be an important determinant of penetration; however, the structure of the polar group can have a dramatic effect. Insulin penetration of mixed lipid films cannot be predicted by the penetration characteristics of the pure components. The possible role of these interactions in determining the geography of the insulin molecule within the liposome and its resultant effects on the stability is discussed.