The anterior cruciate ligament (ACL) can be anatomically divided into anteromedial (AM) and posterolateral (PL) bundles. Current ACL reconstruction techniques focus primarily on reproducing the AM bundle, but are insufficient in response to rotatory loads. The objective of this study was to determine the distribution of in situ force between the two bundles when the knee is subjected to anterior tibial and rotatory loads. Ten cadaveric knees (50+/-10 years) were tested using a robotic/universal force-moment sensor (UFS) testing system. Two external loading conditions were applied: a 134 N anterior tibial load at full knee extension and 15 degrees, 30 degrees, 60 degrees, and 90 degrees of flexion and a combined rotatory load of 10 Nm valgus and 5 Nm internal tibial torque at 15 degrees and 30 degrees of flexion. The resulting 6 degrees of freedom kinematics of the knee and the in situ forces in the ACL and its two bundles were determined. Under an anterior tibial load, the in situ force in the PL bundle was the highest at full extension (67+/-30 N) and decreased with increasing flexion. The in situ force in the AM bundle was lower than in the PL bundle at full extension, but increased with increasing flexion, reaching a maximum (90+/-17 N) at 60 degrees of flexion and then decreasing at 90 degrees. Under a combined rotatory load, the in situ force of the PL bundle was higher at 15 degrees (21+/-11 N) and lower at 30 degrees of flexion (14+/-6 N). The in situ force in the AM bundle was similar at 15 degrees and 30 degrees of knee flexion (30+/-15 vs. 35+/-16 N, respectively). Comparing these two external loading conditions demonstrated the importance of the PL bundle, especially when the knee is near full extension. These findings provide a better understanding of the function of the two bundles of the ACL and could serve as a basis for future considerations of surgical reconstruction in the replacement of the ACL.