Dynamic assessment of three-dimensional (3D) skeletal kinematics is essential for understanding normal joint function as well as the effects of injury or disease. This paper presents a novel technique for measuring in-vivo skeletal kinematics that combines data collected from high-speed biplane radiography and static computed tomography (CT). The goals of the present study were to demonstrate that highly precise measurements can be obtained during dynamic movement studies employing high frame-rate biplane video-radiography, to develop a method for expressing joint kinematics in an anatomically relevant coordinate system and to demonstrate the application of this technique by calculating canine tibio-femoral kinematics during dynamic motion. The method consists of four components: the generation and acquisition of high frame rate biplane radiographs, identification and 3D tracking of implanted bone markers, CT-based coordinate system determination, and kinematic analysis routines for determining joint motion in anatomically based coordinates. Results from dynamic tracking of markers inserted in a phantom object showed the system bias was insignificant (-0.02 mm). The average precision in tracking implanted markers in-vivo was 0.064 mm for the distance between markers and 0.31 degree for the angles between markers. Across-trial standard deviations for tibio-femoral translations were similar for all three motion directions, averaging 0.14 mm (range 0.08 to 0.20 mm). Variability in tibio-femoral rotations was more dependent on rotation axis, with across-trial standard deviations averaging 1.71 degrees for flexion/extension, 0.90 degree for internal/external rotation, and 0.40 degree for varus/valgus rotation. Advantages of this technique over traditional motion analysis methods include the elimination of skin motion artifacts, improved tracking precision and the ability to present results in a consistent anatomical reference frame.