Multilayer protein films in which avidin or streptavidin is used as a cross-linking agent are widely used in bioanalytical laboratories for diagnostics, isolation, and localization. However, comparatively little is known about the structure of these films. In this study, molecular orientation in asymmetric protein bilayers assembled on planar glass substrates was investigated. The multilayer architecture consisted of, in order of deposition, a biotin capped phospholipid bilayer, streptavidin, and yeast cytochrome c biotinylated at cysteine 102. The orientation distribution of the heme groups in the cytochrome c layer was determined using a combination of absorption linear dichroism, measured in a planar integrated optical waveguide-attenuated total reflection geometry, and fluorescence anisotropy, measured in a total internal reflection geometry. A Gaussian model for the orientation distribution was used to recover the mean heme tilt angle and angular distribution about the mean. The orientation distribution for the biotinylated cytochrome c layer was 41 +/-11 degrees, which is nearly identical to the orientation distribution measured previously for yeast cytochrome c covalently immobilized via cysteine 102 to pyridyl-capped phospholipid bilayers.(12) In that case, the cytochrome surface coverage was 0.9 monolayer. In the present study the cytochrome surface coverage was only 0.4 monolayer, which likely precluded lateral interactions between protein molecules. The fact that the orientation distributions were nearly identical shows that lateral interactions are not required to form a macroscopically oriented protein film.