This experimental roentgen stereometric analysis (RSA) study was performed to measure micromotions between the graft and tunnel under submaximal load in anterior cruciate ligament (ACL) reconstruction. The aim was to evaluate the maximum load at failure, linear stiffness, and slippage of bone-patellar-tendon-bone (BPTB) grafts fixed with interference screws compared to quadrupled hamstring grafts fixed with interference screws. We used 15 porcine tibia specimens for the study. In the BPTB group, the 10 x 25-mm bone plugs were fixed in a tunnel with 10 mm in diameter using a 7 x 25-mm titanium interference screw (n = 5) or a biodegradable screw (n = 5). The five hamstring transplants were folded to a four-stranded graft and armed with a baseball stitch suture. The sutured side was fixed with a 7 x 25-mm biodegradable polylactide screw in an 8-mm tunnel. The tibial bones, tendon grafts, and interference screws were marked with tantalum beads. The grafts were mounted to a custom made load frame and loaded parallel to the axis under RSA control increasing the force in steps of 50 N. Micromotions between bone plug, screw, and tibia were measured with RSA. Accuracy of RSA for the in vitro study was evaluated as 0.05 mm. Hamstring grafts failed at significantly lower maximum loads (492 +/- 30 N) than BPTB grafts (658 +/- 98 N). Linear stiffness of the hamstring graft fixations was eight times lower compared to the BPTB grafts (403 +/- 141 N/mm vs 3500 +/- 1300 N/mm). There was no significant difference between the biodegradable and titanium screws in the BPTB group. Slippage of the graft started at 82 +/- 35 N load in the hamstring group and at 428 +/- 135 N in the BPTB group. Slippage of the graft at 75% of the maximum pull-out strength was measured as 0.36 +/- 0.25 mm in the BPTB and 2.58 +/- 1.08 mm in the hamstring group. The interference screw fixation of a quadrupled hamstring graft showed a lower linear stiffness and an earlier slippage compared to a patellar tendon bone plug. Slippage of the hamstring grafts at submaximal loads may result in fixation failure during rehabilitation.