The structure of solution adsorbed and microcontact printed (mu CP) cytochrome c (cyt c) films on glass and indium tin oxide (ITO) was investigated using attenuated total reflectance (ATR) and total internal reflectance fluorescence (TIRF) spectroscopies to determine the orientation of the heme groups in the films. The second and fourth order parameters of the heme as well as information on the angle between the absorption and emission dipoles of the heme, gamma, were experimentally determined. The order parameters of the heme are related to the order parameters of the protein molecule using the known angle between the heme plane and the electrostatic dipole moment of the cyt c protein. The effect of the surface roughness of the substrates (glass and ITO) was also taken into account quantitatively using AFM data. Physically possible order parameters were obtained for the heme group in both solution adsorbed and mu CP films, but not for the electrostatic dipole moment of the protein. In addition, the experimental values of (cos(2) gamma) for immobilized zinc-substituted cyt c are greater than the values of (cos(2) gamma) determined in viscous solutions, which could be an indication that the environment of the heme groups changes upon adsorption. The electron transfer behavior of solution adsorbed and mu CP films on ITO, determined using electrochemical methods, is compared to their orientation distribution and surface coverage as determined by spectroscopic methods.