Anterior cruciate ligament (ACL) reconstruction allows overall good results, but there is still a relevant rate of failure. It is well accepted that the main reason for ACL reconstruction failure is a misplacement of tibial or femoral tunnels. Conventional techniques rely mainly on surgical skill for intra-operative tunnel placement. It has been demonstrated that, even by experienced surgeons, there was a significant variation in the accuracy of tunnel placement with conventional techniques. Navigation systems might enhance the accuracy of ACL replacement. 10 cadaver knees with intact soft-tissue and without any intra-articular abnormalities were studied. We used a non image based navigation system (OrthoPilot ®, Aesculap, Tuttlingen, FRG). Localizers were fixed on bicortical screws on the distal femur and on the proximal tibia. Both cinematic and anatomic registration of the knee joint were performed by moving the knee joint in flexion-extension and palpating relevant intra- and extra-articular landmarks with a navigated stylus. The most anterior, posterior, medial and lateral point of both tibial and femoral attachment of the ACL were marked with metallic pins. The navigated stylus was positioned on these points, and the system recorded its position in comparison to the bone contours. Subsequently, we performed conventional plain AP and lateral X-rays and a CT-scan, and measured the position of the pins in comparison to the bone contours. Finally, all measurements were made again with a caliper after disarticulating the knee joint. We calculated the center of the footprint as the mid-point between the four pins of both tibial and femoral attachment for each measurement technique. All measurements were expressed as percentages of the bone size to compensate for the different sizes. The results were analyzed with a Friedman test with post-hoc comparison, considering the anatomical measurements as the reference. The mean medio-lateral position of the tibial tunnel was (origin at the lateral tibial border): - 52% (SD 2%) for anatomical measurement; - 50% (SD 2%) for navigated measurement; - 49% (SD 2%) for CT measurement; - 51% (SD 2%) for X-ray measurement. The differences were globally significant (p<0.001); there was a significant difference between the reference and navigated and CT measurement techniques. The mean antero-posterior position of the tibial tunnel was (origin at the anterior tibial border): - 53% (SD 7%) for anatomical measurement; - 50% (SD 7%) for navigated measurement; - 50% (SD 7%) for CT measurement; - 50% (SD 7%) for X-ray measurement. The differences were globally significant (p=0.03); there was a significant difference between the reference and the radiological measurement technique. The mean proximo-distal position of the femoral tunnel was (origin at the distal femoral border): - 75% (SD 12%) for anatomical measurement; - 78% (SD 15%) for navigated measurement; - 80% (SD 11%) for CT measurement; - 74% (SD 12%) for X-ray measurement. The differences were globally significant (p=0.05); there was no significant difference between the reference and any measurement technique. The mean antero-posterior position of the femoral tunnel was (origin at the anterior femoral border): - 79% (SD 13%) for anatomical measurement; - 83% (SD 19%) for navigated measurement; - 85% (SD 14%) for CT measurement; - 77% (SD 15%) for X-ray measurement. The differences were globally significant (p<0.01); there was no significant difference between the reference and any measurement technique. Anatomical measurement of the location of the ACL footprint is the gold standard in an experimental situation. CT-scan measurement of the positioning of the ACL replacement tunnels is the gold standard in a clinical situation. X-ray measurement of the positioning of the ACL replacement tunnels is the most used technique in a clinical situation. Navigation might help improving the accuracy and the reproducibility of ACL replacement. However, the precision of each system has to be evaluated. The measurements by the OrthoPilot system were significantly different from the anatomical reference, but the difference appeared to be clinically not relevant. We concluded that this system allowed locating in a precise and accurate way the position of the ACL footprint on both tibial and femoral sides when compared to the reference techniques. This navigation system is precise and accurate for anatomical measurements in the knee joint. This should help the surgeon to define the placement of the tunnels according to anatomical landmarks during ACL replacement. The OrthoPilot navigation system is precise and accurate for anatomical measurement of the tunnel placement during ACL replacement.