A novel X-ray stereoscopy technique, using greyscale information obtained from moving markers, was used to study the 3D motion in both gerbil and rabbit middle ear ossicles in the quasi-static pressure regime. The motion can be measured without visually exposing the ossicles. The ossicles showed non-linear behaviour as a function of both pressure and frequency. For instance, about 80% of the maximum umbo displacement occurs at a 1 kPa (peak-to-peak) pressure load, while a limited increase of the amplitude is noticed when the pressure goes to 2 kPa. In rabbit the ratio of stapes to umbo motion amplitude was 0.35 for a pressure of 2 kPa (peak-to-peak) at 0.5 Hz. From two stereoscopic projections of the marker paths, 3D motion of the ossicles could be calculated. This motion is demonstrated on high-resolution computer models in order to visualize ossicular chain behaviour.