No study has investigated the three-dimensional morphological changes of the anterior cruciate ligament (ACL) during functional activities in vivo. The purpose of this study was to analyze the elongation, rotation (twist), and orientation of the ACL during weight-bearing flexion in five human subjects using dual-orthogonal fluoroscopic images and MR image-based computer models. The ACL consistently decreased in length with flexion. At 90 degrees , the length decreased by 10% compared to its length at full extension. The ACL twisted internally by only 20 degrees at 30 degrees of flexion. The ACL was oriented more vertically (approximately 60 degrees ) and slightly laterally (approximately 10 degrees ) at low flexion angles. These data on in vivo ligament elongation demonstrate that the ACL plays a more important role in lower flexion angles than at higher flexion angles during weight-bearing flexion. These data also suggest that successful ACL reconstruction should not only restore the ligament's elongation behavior, but also its rotational and orientation characteristics, so that normal ACL biomechanics are restored.