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In this paper, a sliding mode control with perturbation estimation (SMCPE) using an extended Kalman filter (EKF) is proposed for the motion tracking control of a micropositioning system with piezoelectric actuation. Based on the solely measurable position information, the EKF on-line estimates not only the velocity but also the hysteresis term, which are used by the SMCPE for real-time control. The feasibility of the proposed control scheme is demonstrated by experimental studies on a XYZ micropositioning stage prototype. Results show that the EKF-based sliding mode control can reduce the hysteresis to a negligible level and lead to a motion tracking with submicron accuracy, which provides a sound base of practical control of the micropositioning system for micro/nano scale manipulation.