A force balance between the ligaments, articular contact, muscles and body weight maintains knee joint stability. Thus, it is important to study anterior cruciate ligament (ACL) biomechanics, in vivo, under weightbearing conditions. Our objective was to compare the ACL strain response under weightbearing and non-weightbearing conditions and in combination with three externally applied loadings: (1) anterior-posterior shear forces, (2) internal-external torques, and (3) varus-valgus moments. A strain transducer was implanted on the ACL of 11 subjects. All joint loadings were performed with the knee at 20 degrees of flexion. A significant increase in ACL strain was observed as the knee made the transition from non-weightbearing to weightbearing. During anterior shear loading, the strain values produced during weightbearing were greater than those of the non-weightbearing knee (shear loads <40N). At higher shear loads, the strain values became equal. During axial torsion, an internal torque of 10Nm strained the ACL when the knee was non-weightbearing while an equivalent external torque did not. Weightbearing significantly increased ACL strain values in comparison to non-weightbearing with the application of external torques and low internal torques (<3Nm). The strains became equal for higher internal torques. For V-V loading, the ACL was not strained in the non-weightbearing knee. However, weightbearing increased the ACL strain values over the range of moments tested. These data have important clinical ramifications in the development of rehabilitation protocols following ACL reconstruction since weightbearing has been previously thought to provide a protective mechanism to the healing graft.