This study aims at developing an accurate method for analyzing in vivo knee kinematics using single plane fluoroscopy and 3D bone models. The study was carried out on 3 porcine knees. Three sphere markers were fixed to both femur and tibia. A CT scan was acquired for each bone to create a 3D bone model. After being CT scanned, the femur and tibia were fixed using an external fixation device, resembling anatomical positions of knee. Ten single plane fluoroscopic images of 3 sets of fixed bones were obtained with different flexion angles and different directions of image acquisition. Digitally reconstructed radiographs (DRRs) were generated from 3D bone volume models by a voxel projection technique. The relative 3D-pose (full 6 degrees-of-freedom parameters) between femur and tibia were determined by matching the DRR of each bone model with the fluoroscopic image by maximizing the similarity measure between the 2 images. The true value of the relative pose was measured by a 3D coordinate measuring machine. The mean errors of the 3 rotation parameters were within 0.5 degrees. For the translation parameters the mean error took its maximal value of 1.5 mm in the out-of-plane direction. The result obtained shows a potential of the proposed method for the accurate analysis of in vivo knee kinematics.