The purpose of the present study is to develop a direct and accurate method for measuring knee kinematics by using single-plane fluoroscopy. The study was carried out on a human cadaver femur. Computed tomography (CT) scan data of the femur was taken in order to construct 3D bone volume model of the femur. The femur was placed on an acrylic holder that was attached to a micromanipulator. The femur was rotated about in each orthogonal axis of the micromanipulator over a range of ±2° in 1° increments and then translated along each orthogonal axis over a range of ±2 mm in 1-mm increments. The 3-dimensional (3D) position of the femur (in other words, the 6 degree-of-freedom (DOF) parameters) was recovered by matching the digitally reconstructed radiographs (DRRs) generated from the 3D volume model of the femur and single-plane fluoroscopic images taken from the 25 positions generated by using the micromanipulator. The root-mean-square error (RMSE) of the overall rotation parameters was within 1.4°. For the translation parameters RMSE took its maximal value of 7.8 mm in the out-of-plane direction. This indicates that the present method has potential for clinical application. Copyright © 2009 by The Japan Society of Mechanical Engineers.