The objective of the current study was to use fluoroscopy to accurately determine the three-dimensional (3D), in vivo, weight-bearing kinematics of 10 normal and five anterior cruciate ligament deficient (ACLD) knees. Patient-specific bone models were derived from computed tomography (CT) data. 3D computer bone models of each subject's femur, tibia, and fibula were recreated from the CT 3D bone density data. Using a model-based 3D-to-2D imaging technique registered CT images were precisely fit onto fluoroscopic images, the full six degrees of freedom motion of the bones was measured from the images. The computer-generated 3D models of each subject's femur and tibia were precisely registered to the 2D digital fluoroscopic images using an optimization algorithm that automatically adjusts the pose of the model at various flexion/extension angles. Each subject performed a weight-bearing deep knee bend while under dynamic fluoroscopic surveillance. All 10 normal knees experienced posterior femoral translation of the lateral condyle and minimal change in position of the medial condyle with progressive knee flexion. The average amount of posterior femoral translation of the lateral condyle was 21.07 mm, whereas the average medial condyle translation was 1.94 mm, in the posterior direction. In contrast, all five ACLD knees experienced considerable change in the position of the medial condyle. The average amount of posterior femoral translation of the lateral condyle was 17.00 mm, while the medial condyle translation was 4.65 mm, in the posterior direction. In addition, the helical axis of motion was determined between maximum flexion and extension. A considerable difference was found between the center of rotation locations of the normal and ACLD subjects, with ACLD subjects exhibiting substantially higher variance in kinematic patterns.