An increase in anterior laxity following reconstruction of the anterior cruciate ligament (ACL) can result from lengthening of the graft construct either at the sites of fixation and/or between the sites of fixation (i.e., graft substance). Roentgen stereophotogrammetric analysis (RSA), which requires that radio-opaque markers be attached to the graft, has been shown to be a useful technique in determining lengthening in these regions. Previous methods have been used for attaching radio-opaque markers to the graft, but they all have limitations particularly for single-loop grafts. Therefore, the objective of this study was to evaluate injecting markers directly into the substance of a tendon as a viable method for measuring lengthening of single-loop graft constructs by determining the maximum amount of migration after cyclic loading. Tantalum spheres of 0.8 mm diameter were used as tendon markers. Ten single-loop tendon grafts were passed through tibial tunnels drilled in calf tibias and fixed with a tibial fixation device. Two tendon markers were inserted in one tendon bundle of each graft and the grafts were cyclically loaded for 225,000 cycles from 20 N to 170 N. At specified intervals, simultaneous radiographs were obtained of the tendon markers. Marker migration was computed as the change in distance between the two tendon markers parallel to the axis of the tibial tunnel. Marker migration had a root mean square (RMS) value of less than 0.1 mm. Because the RMS value indicates the error introduced into measurements of lengthening and because this error is negligible, the method described for attaching markers to single-loop ACL grafts has the potential to be useful for determining lengthening of single-loop ACL graft constructs in in vivo studies in humans.