Three different methods for preparing cytochrome c (cyt c) films on planar indium tin oxide (ITO) electrodes have been characterized and compared using electrochemical and surface spectroscopy methods. Protein films formed by adsorption of dissolved cyt c and by microcontact printing with both hydrophobic and hydrophilic poly(dimethylsiloxane) (PDMS) stamps are compared in terms of the total and electrochemical surface coverage, standard reduction potential, and rate of direct electron transfer with the ITO surface. Plasma treatment. of the PDMS stamps, which renders them hydrophilic, dramatically increases the surface coverage of printed films to a level comparable to that of films formed by adsorption of dissolved cyt c. Their electrochemical properties are also very similar. This work is the first report of the direct electrochemical activity in a metalloprotein film formed by microcontact printing on an electrode surface.