Recent reports have indicated that anatomical placement of the anterior cruciate ligament (ACL) graft is an important factor for restoration of joint function following ACL reconstruction. The objective of this study was to address a need for a better understanding of anatomical variations in ACL position and orientation within the joint. Specifically, variations in the ACL anatomy were assessed by testing for side-to-side ACL footprint location symmetry in a healthy population relative to the operative and contralateral knee in a patient population after traditional transtibial single-bundle ACL reconstruction. MRI and three-dimensional modeling techniques were used to determine the in vivo tibiofemoral ACL footprint centers and the resulting ACL orientations in both knees of 30 healthy subjects and 30 subjects after transtibial ACL reconstruction. While there were substantial inter-subject variations in ACL anatomy, the side-to-side RMS differences in the ACL footprint center were 1.20 and 1.34 mm for the femur and tibia, respectively, for the healthy subjects and no clinically meaningful intra-subject differences were measured. However, there were large intrasubject side-to-side differences after transtibial ACL reconstruction, with ACL grafts placed 5.63 and 7.64 mm from the center of the contralateral femoral and tibial ACL footprint centers, respectively. Grafts were placed more medial, anterior, and superior on the femur and more posterior on the tibia; producing grafts that were more vertical in the sagittal and coronal planes. Given the large variation among subjects, these findings advocate the use of the contralateral ACL morphology for retrospectively evaluating patientspecific anatomic graft placement. ß