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Background: There is controversy regarding the functional role of the posterolateral (PL) bundle of the anterior cruciate ligament (ACL). Purpose: To evaluate the in vivo function of the PL and anteromedial (AM) bundles of the ACL during anatomic double-bundle (DB) ACL reconstruction for acute, isolated ACL tears utilizing a computer navigation system to track intraoperative knee kinematics. Study Design: Controlled laboratory study. Methods: Fifteen patients with acute, isolated ACL tears who underwent anatomic DB ACL reconstruction formed the sample for this study. During surgery, knees were examined by a clinician preoperatively, after fixation of the PL bundle, and after fixation of both the PL and AM bundles. An image-free computer navigation system with custom-made software recorded the data during kinematic tests. The examination consisted of the Lachman and anterior drawer tests, internal-external rotation at 30° of knee flexion, and varus-valgus rotation at 30° of knee flexion. Paired Wilcoxon tests were performed to evaluate the effects of each bundle. The comparisons included ACL-deficient to PL bundle–reconstructed knees, ACL-deficient to DB ACL–reconstructed knees, and PL bundle–reconstructed to DB ACL–reconstructed knees. Significance was set at P<.017 to account for the multiple comparisons. Results: Fixation of the PL bundle significantly improved knee laxity during the Lachman and anterior drawer tests as well as internal-external rotation at 30° of knee flexion (P<.017 for all tests). The addition of the AM bundle further improved knee laxity during the Lachman and anterior drawer tests compared with PL bundle fixation as well as during varus-valgus rotation at 30° of knee flexion compared with ACL-deficient knees (P<.017 for all tests). Conclusion: This in vivo study demonstrates that isolated PL bundle reconstruction improves laxity in an ACL-deficient knee and that the addition of the AM bundle improves laxity parameters further. Clinical Relevance: Abnormal knee kinematics is known to be linked to the earlier onset of osteoarthritis and lower rates of return to sport. This study suggests that both the AM and PL bundles are important to stabilize ACL-deficient knees. [ABSTRACT FROM AUTHOR]