The purpose of this study was to investigate in vivo three- dimensional tibiofemoral kinematics and femoral condylar motion in knees with anterior cruciate ligament (ACL) deficiency during a knee bend activity. Ten patients with unilateral ACL rupture were enrolled. Both the injured and contralateral normal knees were imaged using biplane radiography at extension and at 15°, 30°, 60°, 90°, and 120° of flexion. Bilateral knees were next scanned by computed tomography, from which bilateral three-dimensional knee models were created. The in vivo tibiofemoral motion at each flexion position was reproduced through image registration using the knee models and biplane radiographs. A joint coordinate system containing the geometric center axis of the femur was used to measure the tibiofemoral motion. In ACL deficiency, the lateral femoral condyle was located significantly more posteriorly at extension and at 15° (p < 0.05), whereas the medial condylar position was changed only slightly. This constituted greater posterior translation and external rotation of the femur relative to the tibia at extension and at 15° (p < 0.05). Furthermore, ACL deficiency led to a significantly reduced extent of posterior movement of the lateral condyle during flexion from 15° to 60° (p < 0.05). Coupled with an insignificant change in the motion of the medial condyle, the femur moved less posteriorly with reduced extent of external rotation during flexion from 15° to 60° in ACL deficiency (p < 0.05). The medial- lateral and proximal-distal translations of the medial and lateral condyles and the femoral adduction-abduction rotation were insignificantly changed after ACL deficiency. The results demonstrated that ACL deficiency primarily changed the anterior-posterior motion of the lateral condyle, producing not only posterior subluxation at low flexion positions but also reduced extent of posterior movement during flexion from 15° to 60°.