Recently, there has been a tremendous impetus on anatomical reconstruction of the anterior cruciate ligament (ACL), and the double-bundle reconstruction concept has been advocated by many authors. It is, therefore, important to understand how the lengths of the two bundles of the ACL vary during different knee flexion angles as this could influence the angle of graft fixation during surgery. The aim of this study is to determine the change in length of the ACL bundles during different knee flexion angles. Ten subjects with normal knees were evaluated. A high-resolution computer tomography scan was performed, and 3D knee images were obtained. These images were then imported to customized software, and digital length measurement of four virtual bundles (anatomical single bundle, AM, PL and over the top) was evaluated from fixed points on the femur and tibia. Length-versus-flexion curves were drawn, and statistical analysis was performed to evaluate changes in length for each bundle at varying angles of knee flexion (0 degrees, 45 degrees, 90 degrees and 135 degrees). All virtual bundles achieved greatest lengths at full extension. There was a significant difference between the posterolateral bundle length when compared to the other bundles at full extension. There were no significant differences between the lengths of the anteromedial and the over the top single bundles at all angles of knee flexion. Three-dimensional computer tomography can be used to assess the length changes of the virtual anterior cruciate ligament bundles, thereby allowing a better understanding of bundle function in clinical situations.