The vibration of the hearing organ that occurs during sound stimulation is based on mechanical interactions between different cellular structures inside the organ of Corti. The exact nature of these interactions is unclear and subject to debate. In this study, dynamic structural changes were produced by stepwise alterations of scala tympani pressure in an in vitro preparation of the guinea pig temporal bone. Confocal images were acquired at each level of pressure. In this way, the motion of several structures could be observed simultaneously with high resolution in a nearly intact system. Images were analyzed using a novel wavelet-based optical flow estimation algorithm. Under these conditions, the reticular lamina moved as a stiff plate with a center of rotation in the region of the inner hair cells. Despite being enclosed in several types of supporting cells, the inner hair cells, together with the adjacent inner pillar cells, moved in a manner signifying high compliance. The outer hair cells displayed radial motion indicative of cellular bending. Together, these results show that shearing motion occurs between several parts of the organ, and that structural relationships within the organ change dynamically during displacement of the basilar membrane.