Purpose: To evaluate dynamic three-dimensional (3D) kinematic properties of the anterior cruciate ligament (ACL)-insufficient knees and healthy contralateral knees in awake patients during the Lachman test using biplane fluoroscopy. Materials and Methods: Ten patients with unilateral ACL-insufficient knees and healthy contralateral knees were enrolled in this study. Each patient underwent the Lachman test three times in the awake state. The knee joint motions were captured using biplane fluoroscopy. After manual registration of 3D surface data from 3D-computed tomography to biplane images, dynamic 3D kinematic data were analyzed. Results: The average anteroposterior (AP) translation of the medial femoral epicondyle of the ACL-insufficient knees (11.5±4.2 mm) was significantly greater than that of the contralateral knees (7.7±3.5 mm) (p<0.05). However, there was no statistically significant side-to-side difference in the average AP translation of the lateral femoral epicondyle. During the Lachman test, the distal femur was more externally rotated than the proximal tibia, which showed significant difference between both sides.Conclusions: During the Lachman test, the medial femoral epicondyle of the ACL-insufficient knee exhibited greater AP motion than that of the contralateral knee, whereas there was no significant side-to-side difference with regard to the AP motion of the lateral femoral epicondyle.