We have developed a marker-based 3D motion capture system for high-precision quantification of mouse locomotion. In our previous studies, we observed that a low dose of a synthetic agonist of cannabinoid CB1 and CB2 receptors, CP55,940 (0.3 mg/kg), causes a small reduction in general locomotion during open-field exploration, but does not inhibit general locomotion during vertical climbing. However, we observed that it induces significant changes in step kinematics during both tasks, which were more pronounced during climbing.We used adult male C57BL/6 mice in a within-subject, randomized design (n=6-10). We recorded voluntary climbing behavior on a spoked mesh wheel, in mice treated with vehicle (1:1:18 EtOH, Kolliphor, saline), a low dose of CP55,940 (0.3 mg/kg), or harmaline (20 mg/kg, i.p. 30 min pretreatment). Mice were implanted with permanent markers located on the hips, shoulder blades, hindlimb knees, and ankles. A high-speed, high-resolution 3D motion capture system (Qualisys) was used to track 3D trajectories and velocity of markers during voluntary locomotor climbing task.Harmaline, at 20 mg/kg, induces strong whole-body tremor and was found to significantly inhibit locomotion during climbing and reduce the total number of steps. Moreover, the mean swing of the ankle was significantly shorter in distance and the average speed of the swing was reduced. CP55,940 did not reduce general locomotion but affected step kinematics, decreasing step height and speed, which is consistent with our previous observations. In conclusion, marker-based motion capture is a precise method to assess 3D locomotion and step kinematics in mice.