Aims: Serial dilation of the walls of the bone tunnel has been advocated to provide more dense bone-tunnel walls and optimal conditions for rigid fixation in anterior cruciate ligament (ACL) reconstruction with soft tissue grafts. The aim of this study was to compare the initial fixation strength obtained using serial dilation versus conventional extraction drilling in ACL reconstruction. Methods: Initial strength of doubled anterior tibialis tendon grafts fixed with bioabsorbable interference screw was assessed in 21 pairs of human cadaver tibiae. Bone tunnels were created with either serial dilation or conventional extraction drilling (cannulated drill bits). The specimens were subjected to a cyclic-loading test (1500 loading cycles between 50 and 200 N at 0.5 Hz frequency). The specimens surviving the cyclic-loading test were loaded to failure at a rate of 1.0 m/min (single-cycle load-to-failure test). Results: During the cyclic-loading test, no significant stiffness or displacement differences were observed between the two bone-tunnel techniques. Three specimens failed in the serial-dilation group, while there were six failures in the extraction-drilling group. In the subsequent single-cycle load-to-failure test, the average yield load was 473 ± 110 N for the serial-dilation group and 480 ± 115 N for the extraction-drilling group (P=0.97). No significant difference between the two bone-tunnel techniques was found with regard to stiffness nor mode of failure. Conclusions: Serial dilation of the bone-tunnel walls does not increase the initial fixation strength of soft tissue grafts in ACL reconstruction.