Instrumented tibiofemoral (bone-to-bone) excursion wires were implanted in the mid-substance of the anteromedial, central, and posterior fiber-regions of the anterior cruciate ligament through limited anterior and posterior arthrotomies in eight fresh knees from cadavera. The change in the distance of linear separation between each pair of osseous fiber-insertion sites was measured and was plotted against the angle of flexion of the knee as the knee was cycled through a 120-degree range of motion. Testing conditions likely to be present during intraoperative testing for isometry were used (anterior cruciate fibers transected, quadriceps relaxed, femur stabilized with the patient in the supine position and the leg freely dependent, and motion of the knee induced in neutral rotation by force applied at the level of the foot). In no instance did the insertion-site centers of any fiber-region exhibit isometric behavior (change in the distance of linear separation of 1.0 millimeter or less). The least deviations from isometry (range, 1.4 to 3.1 millimeters) were observed for the anteromedial sites, under conditions when the gravitational dependency of the lower leg was constrained. When the leg hung in a dependent manner during passive motion, the deviation from isometry of the anteromedial sites of insertion increased significantly (range, 2.8 to 5.6 millimeters). The central sites of insertion were generally less isometric than the anteromedial sites, and the posterior sites were the least isometric, regardless of testing conditions.