A decrease of deep knee flexion torque after anterior cruciate ligament (ACL) reconstruction, using a semitendinosus (and gracilis) tendon, has been reported. However, the cause of this weakness remains controversial. Architectural and functional differences in the knee flexor muscles influence this weakness. the fiber length of the semitendinosus, gracilis, semimembranosus, and biceps femoris were directly measured in six human cadavers. The flexion torque and EMG of the hamstrings were measured in both limbs of 16 patients (23+/-5 years) after ACL reconstruction (12-43 months post-operation), using ipsilateral semitendinosus tendon. Magnetic resonance imagings were taken, over both the thighs of those patients, to measure muscle volume and to confirm a state of semitendinosus tendon regeneration. The position of the musculotendinous junction of the semitendinosus was also analyzed. The fiber length of the semitendinosus and gracilis were three to four times longer than that of the semimembranosus and biceps femoris. The difference of flexion torque between the normal and ACL reconstructed limbs significantly increased as the knee flexion angle increased. The EMG value for the semimembranosus and biceps femoris of both limbs as well as the semitendinosus of the ACL reconstructed limbs, significantly reduced as the knee flexion angle was increased. The volume of the semitendinosus in the reconstructed limb was significantly smaller than in normal limbs. The regeneration of the semitendinosus tendon was confirmed in all subjects, and the musculotendinous junction position of the reconstructed limb in almost all subjects was found in further image from the knee joint space than that for the normal limb. The decrease of deep knee flexion torque, after ACL reconstruction, could be due to the atrophy and shortening of the semitendinosus after its tendon has been harvested, as well as the lack of compensation from the semimembranosus and biceps femoris, due to the architectural differences between the semitendinosus and the semimembranosus and biceps femoris.