Electron spin resonance (ESR) anisotropy at 10 K and electronic absorbance linear dichroism (LD) at room temperature were used to establish the orientation of the protein myoglobin (Mb) in cast films of : phosphatidylcholines, didodecyldimethylammonium bromide, and dihexadecyl phosphate. The membranes feature stacked lipid bilayer-like structures as suggested by the similarity of gel-to-liquid crystal phase transition temperatures of films with those of bilayer vesicles of the same amphiphile. ESR and LD gave good agreement for the angle of orientation between the Mb heme plane and the film normal, with values ranging between 55 degrees and 61 degrees. Weak dependence of Mb orientation on the type of head group and phase state of these films suggests that a significant fraction of the protein may be imbedded in hydrophobic lipid bilayer regions. Results suggest that the films are organized by self-assembly driven by intermolecular interactions. The highest degree of order was found in Mb-DDAB films, and results suggested more disorder in films of the other amphiphiles.