Intercalation of the heme proteins, cytochrome c (Cyt c) and bovine hemoglobin in thermally evaporated fatty lipid films under protein-friendly conditions by a novel beaker-based diffusion mechanism is described. An attractive electrostatic interaction between charged groups on the protein surface and ionized lipid molecules in the film is primarily responsible for the diffusion of the proteins from the solution into the lipid matrix. To highlight the generality of the approach, Cyt c (cationic at a pH of ca. 7) and hemoglobin (anionic at pH = 9) have been incorporated into thermally evaporated arachidic acid (anionic at pH = 7) and octadecylamine (cationic at pH = 9) films, respectively, by simple immersion of the films in the respective protein solutions. Quartz crystal microgravimetry measurements Were used to follow the kinetics of protein diffusion into the films and was found to be strongly dependent on the protein solution pH. The electrostatic nature of coordination between the proteins and matrix lipid molecules enables reversal of the protein adsorption process-the proteins could be "pumped out" of the composite films into solution under appropriate conditions. Fluorescence spectroscopy and Fourier transform infrared spectroscopy studies indicated little perturbation to the native protein structure, while UV-Vis spectroscopy was used to follow the redox behavior of Cyt c in arachidic acid films. Incorporation of Cyt c in the lipid matrix leads to a lamellar film structure, with a repeat distance of 58.8 A. The advantages of this approach over other methods currently used for entrapment of proteins is briefly discussed.