Molecular orientation in films of yeast cytochrome c immobilized via disulfide bonding between cysteine 102 and the thiol tail groups of self-assembled monolayers (SAMs) coated on planar glass substrates was investigated. The orientation distribution of the heme groups in the protein film was determined using a combination of absorption linear dichroism, measured in a planar integrated optical waveguide-attenuated total reflection geometry, and emission anisotropy, measured in a total internal reflection fluorescence geometry. The mean heme tilt angle and angular distribution about the mean were recovered using a Gaussian model for the orientation distribution. These data are the first orientation distribution measurements reported for a protein film immobilized using a site-directed bonding strategy. The results show that the molecular architecture examined in this study does not produce a highly oriented protein film. A significant fraction of the immobilized cytochrome c is nonspecifically adsorbed to the SAM surface, which produces a relatively broad distribution of heme orientations.