Although single-loop tibialis tendon allografts have increased in popularity owing to their many advantages over patellar tendon and double-loop hamstring tendon autografts, some percentage of the patient population do not have clinically stable knees following anterior cruciate ligament reconstruction with single-loop tibialis tendon allografts. Therefore, it would be advantageous to determine the causes of increased anterior laxity which ultimately must be traced to lengthening of the graft construct. One objective of this study was to demonstrate the feasibility of using Roentgen stereophotogrammetric analysis (RSA) to determine the causes of lengthening of a single-loop graft construct subjected to cyclic loading. A second objective was to determine which cause(s) contributes most to an increase in length of this graft construct. Radio-opaque markers were inserted into ten grafts to measure the lengthening at the sites of the tibial and femoral fixations and between the sites of fixation. Each graft was passed through a tibial tunnel in a calf tibia, looped around a rigid cross-pin, and fixed to the tibia with a Washerloc fixation device. The grafts were cyclically loaded for cycles from . Prior to and at intervals during the cyclic loading, simultaneous radiographs were taken. RSA was used to determine the three-dimensional coordinates of the markers from which the lengthening at the sites of fixation and between the sites of fixation was computed at each interval. The sites of the femoral and tibial fixations were the largest contributors to the increase in length of the graft construct, with maximum average values of 0.68 and mm, respectively, after cycles. The graft substance between the sites of fixation contributed least to lengthening of the graft, with a maximum average value of mm. Ninety percent of the maximum average values occurred before cycles of loading for the largest contributors. RSA proved to be a useful method for measuring lengthening due to all three causes. Lengthening of the graft construct at the sites of both fixations is sufficiently large that the combined contributions may manifest as a clinically important increase in anterior laxity.