Abstract An in vitro study of eight cadaveric knees was conducted to investigate the effect of initial graft tension on the laxity and full three-dimensional kinematics of the anterior cruciate ligament reconstructed knee. A parallel strand, prototype, expanded polytetrafluoroethylene graft (W. L. Gore and Associates, Flagstaff, AZ, U.S.A.) was used. The graft was placed in the over-the-top position with initial tensions of 18, 36, 54, 72, and 90 N applied with the knee in full extension or at 30° of flexion. The motion of the tibia relative to the femur was measured by a 6°-of-freedom spatial linkage, and the applied forces and moments, the quadriceps force, and the graft tension were measured by load cells. Near normal anterior laxity in the Lachman test was restored with all the tested initial graft tensions. However, over constraint, posterior, lateral, and external tibial subluxation, and abnormalities in joint stiffness developed as the initial graft tension increased. Graft tension-related posterior tibial subluxation resulted in an increase in quadriceps force needed to achieve full extension.