INTRODUCTION. The restoration of physiological kinematics is one of the goals of a total knee arthroplasty (TKA). Navigation systems have been developed to allow an accurate and precise placement of the implants. But its application to the intraoperative measurement of knee kinematics has not been validated. The hypothesis of this study was that the measurement of the knee axis, femoral rotation, femoral translation with respect to the tibia, and medial and lateral femorotibial gaps during continuous passive knee flexion by the navigation system would be different from that by fluoroscopy taken as reference. MATERIAL – METHODS. Five pairs of knees of preserved specimens were used. The e.Motion FP ® TKA (B-Braun Aesculap, Tuttlingen, Germany) was implanted using the OrthoPilot TKA 4.3 version and Kobe version navigation system (B-Braun Aesculap, Tuttlingen, Germany). Kinematic recording by the navigation system was performed simultaneously with fluoroscopic recording during a continuous passive flexion-extension movement of the prosthetic knee. Kinematic parameters were extracted from the fluoroscopic recordings by image processing using JointTrack Auto ® software (University of Florida, Gainesville, USA). The main criteria were the axis of the knee measured by the angle between the center of the femoral head, the center of the knee and the center of the ankle (HKA), femoral rotation, femoral translation with respect to the tibia, and medial and lateral femorotibial gaps. The data analysis was performed by a Kappa correlation test. The agreement of the measurements was assessed using the intraclass correlation coefficient (ICC) and its 95% confidence interval. RESULTS. The respective CCIs were as follows: HKA angle 0.839 [0.820; 0.856]; femoral translation 0.560 [0.517; 0.600]; femoral rotation 0.652 [0.616; 0.686]; medial femorotibial gap 0.905 [0.894; 0.916]; lateral femorotibial gap 0.767 [0.740; 0.791]. DISCUSSION. Measurements of TKA kinematics by the navigation system and by fluoroscopy were consistent for HKA angle and medial and lateral femorotibial gaps, but not for femoral translation and femoral rotation. These differences can be explained by a methodological bias. At the end of this work, the specific navigation system cannot be considered as a reliable instrument for measuring the kinematics of a TKA