The ACL-deficient dog is a model for investigating the development and progression of mechanically driven osteoarthrosis of the knee. ACL loss creates dynamic instability in the ACL-deficient knee which presumably leads to progressive joint degeneration, but the nature of this instability over the time course of disease development is not well understood. The goal of this study was to characterize three-dimensional motion of the canine knee during gait, before and serially for two years after ACL transection. Canine tibial-femoral kinematics were assessed during treadmill gait before and serially for two years after ACL transection (ACL-D group; 18 dogs) or sham transection (ACL-I group; five dogs). Kinematic data was collected at 250 frames/s using a biplane video-radiographic system. Six degree-of-freedom motions of the tibia relative to the femur were calculated, and values immediately prior to pawstrike as well as the maximum, minimum, midpoint and range of motion during early/mid stance were extracted. Between-group differences relative to baseline (pre-transection) values, as well as changes over time post-transection, were determined with a repeated-measures ANCOVA. In the ACL-D group, peak anterior tibial translation (ATT) increased by 10 mm (p < 0.001), and did not change over time (p=0.76). Pre-pawstrike ATT was similar to ACL-intact values early on (2-4 months) but then increased significantly over time, by 3.5 mm (p < 0.001). The range of ab/adduction motion nearly doubled after ACL loss (from 3.3 degrees to 6.1 degrees). The magnitude (midpoint) of knee adduction also increased significantly over time (mean increase 3.0 degrees; p = 0.036). All changes occurred primarily between 6 and 12 months. There were no significant differences between groups in the transverse plane, and no significant changes over time in the ACL-I group. In summary, peak anterior tibial translation and coronal-plane instability increased immediately after ACL loss, and did not improve with time. ATT just prior to pawstrike and mean knee adduction throughout stance became progressively more abnormal with time, with the greatest changes occurring between 6 and 12 months after ACL transection. This may be due to overload failure of secondary restraints such as the medial meniscus, which has been reported to fail in a similar timeframe in the ACL-deficient dog. The relationships between these complex mechanical alterations and the rate of OA development/progression are currently under investigation.