AIM To quantitatively assess rotatory and anterior-posterior instability in vivo after anterior cruciate ligament (ACL) reconstruction using bone-patellar tendon-bone (BTB) autografts, and to clarify the influence of tunnel positions on the knee stability. METHODS Single-bundle ACL reconstruction with BTB autograft was performed on 50 patients with a mean age of 28 years using the trans-tibial (TT) (n = 20) and trans-portal (TP) (n = 30) techniques. Femoral and tibial tunnel positions were identified from the high-resolution 3D-CT bone models two weeks after surgery. Anterolateral rotatory translation was examined using a Slocum anterolateral rotatory instability test in open magnetic resonance imaging (MRI) 1.0-1.5 years after surgery, by measuring anterior tibial translation at the medial and lateral compartments on its sagittal images. Anterior-posterior stability was evaluated with a Kneelax3 arthrometer. RESULTS A total of 40 patients (80%) were finally followed up. Femoral tunnel positions were shallower (P &lt
0.01) and higher (P &lt
0.001), and tibial tunnel positions were more posterior (P &lt
0.05) in the TT group compared with the TP group. Anterolateral rotatory translations in reconstructed knees were significantly correlated with the shallow femoral tunnel positions (R = 0.42, P &lt
0.01), and the rotatory translations were greater in the TT group (3.2 ± 1.6 mm) than in the TP group (2.0 ± 1.8 mm) (P &lt
0.05). Side-to-side differences of Kneelax3 arthrometer were 1.5 ± 1.3 mm in the TT, and 1.7 ± 1.6 mm in the TP group (N.S.). Lysholm scores, KOOS subscales and reinjury rate showed no difference between the two groups. CONCLUSION Anterolateral rotatory instability significantly correlated shallow femoral tunnel positions after ACL reconstruction using BTB autografts. Clinical outcomes, rotatory and anterior-posterior stability were overall satisfactory in both techniques, but the TT technique located femoral tunnels in shallower and higher positions, and tibial tunnels in more posterior positions than the TP technique, thus increased the anterolateral rotation. Anatomic ACL reconstruction with BTB autografts may restore knee function and stability.