Purpose: Our goal was to characterize the type of biologic anchor of hamstring tendons to the femoral tunnel in cases of transfixion fixation for the anterior cruciate ligament (ACL) reconstruction. The histologic bone-hamstring tendon anchorage is not yet clearly understood despite many experimental and some clinical studies. It constitutes the weak point of the ACL reconstruction. The type of fixation, either distant from the joint such as transfixion fixation or at the tunnel entrance such as aperture fixation will determine a specific tendon-bone healing process. Type of study: Histological study. Methods: We performed ACL reconstruction with 4 strands of semitendinosus and gracilis tendons fastened by a transfixion fixation. Femoral fixation was secured by transfixion (Transfix; Arthrex, Naples, CA) and tibia fixation by a biodegradable interference screw and 2 staples. Between 3 and 20 months after surgery, we performed 12 hamstring tendon biopsies (in 9 men and 3 women; mean age, 29 years). Biopsies were performed 2 cm from the femoral outlet in 10 patients undergoing hardware removal or by coring the femoral tunnel in 2 cases of repeat rupture. In 8 cases, the femoral device was removed for persistent lateral pain, in 2 cases for instability of the hardware, and in 2 cases a repeat rupture of the graft occurred. The samples were taken by coring a tunnel 5 mm in diameter, with a tubular harvester, along the femoral Transfix axis. Each fragment was stained with H&E, Solochrome cyanine, or Masson-trichrome, and microscopical examination was performed, including polarized light. Results: At 3 months (in 1 case), a fibrovascular interface was seen between the tendon and uncalcified osteoid with very few collagen fibers. At 5 and 6 months (in 2 cases), some Sharpey-like fibers and less immature woven bone was seen. Maturity of the secondary insertion was seen after at least 10 months in 5 cases. In 2 cases, no contact was seen at the biopsy site despite good clinical stability. The 2 remaining cases underwent repeat rupture at the midsubstance of the graft at 12 and 17 months after surgery. In the first case, the tendon-bone fixation was limited at the outlet of the femoral tunnel with no fixation inside the tunnel. In the second case, the fixation was continuous with Sharpey fibers along the tunnel. Conclusions: According to our histologic results in patients, the time to obtain a mature indirect anchorage at the top of the tunnel was 10 to 12 months, which is much longer than in reported animal models (6 to 24 weeks). To our knowledge, this is the first clinical study reporting the histologic type of femoral ligament insertion 2 cm from the outlet of the tunnel with hamstring autograft for ACL reconstruction.