The in situ forces and their distribution within the human anterior cruciate ligament (ACL) can clarify this ligament's role in the knee and help to resolve controversies regarding surgical treatment of ACL deficiency. We used a universal force-moment sensor (UFS) to determine the magnitude, direction, and point of application of the in situ forces in the ACL in intact human cadaveric knees. Unlike previous studies, this approach does not require surgical intervention, the attachment of mechanical devices to or near the ACL, or a priori assumptions about the direction of in situ force. Anterior tibial loads were applied to intact knees, which were limited to 1 degree of freedom at 30 degrees flexion. The in situ forces developed in the ACL were lower than the applied force for loads under 80 N, but larger for applied loads of more than 80 N. The direction of the force vector corresponded to that of the anteromedial (AM) portion of the ACL insertion on the tibial plateau. The point of force application was located in the posterior section of the anteromedial portion of the tibial insertion site. The anterior and posterior aspects of the anteromedial portion of the ACL supported 25% and 70% of the in situ force, respectively, with the remainder carried by the posterolateral portion. We believe that the data obtained with this new UFS methodology improves our understanding of the role of the ACL in knee function, and that this methodology can be easily extended to study the function of other ligaments.