The tectorial membrane (TM) is a highly hydrated non-cellular matrix situated over the sensory cells of the cochlea. It is widely accepted that the mechanical coupling, between the TM and outer hair cells stereocilia bundles, plays an important role in the cochlea energy transduction mechanism. Recently, we provided supporting evidence for the existence of mechanical coupling by demonstrating that the mechanical properties of the TM change along its longitudinal direction. Since the biochemical composition of the TM is similar throughout its entire length, it is likely that structural differences induce the observed material properties changes. Presently, however, the structure of the TM under physiological environments remains unknown. In this work, the 3D structure of native TM samples is shown by using two-photon second-harmonic imaging microscopy. We find that the collagen fibers at the basal region are arranged in a parallel orientation while being tilted in an angle with respect to the plane of the TM surface at the apical region. Moreover, we find an intensified marginal band at the basal OHC zone which forms a shell-like structure which engulfs the stereocilium imprints surface of the TM. In supports of our previous mechanical characterization, the analysis presented here provides a structural basis for the changes in TM's mechanical properties. (c) 2007 Elsevier Inc. All rights reserved.