The effects of functional load and muscle force application on isometry of the posterior cruciate ligament were determined. Eight fresh-frozen cadaver knees were mounted in a custom-designed rig. A full range of motion and muscle forces were applied through the quadriceps, hamstring, and gastrocnemius tendons during a simulated static squat maneuver. The low-load isometric posterior cruciate ligament point was located 5.63 mm proximal and 0.18 mm anterior to the anatomic center of the posterior cruciate ligament origin on the femur. The high-load state, with no gastrocnemius and hamstring muscle forces applied, shifted the isometric point 6.32 mm proximal and 6.72 mm anterior (P < 0.05). Loading the hamstring and gastrocnemius muscles also shifted the isometric point (P < 0.05). This study indicated that the most isometric region of the posterior cruciate ligament femoral attachment changed significantly when functional loads and muscle forces were applied to the knee. This finding may have implications for both surgical reconstruction and rehabilitation of the posterior cruciate ligament-injured knee.