Abstract Background In this paper, we present a new method for the calibration of a microscope and its registration using an active optical tracker. Methods Practically, both operations are done simultaneously by moving an active optical marker within the field of view of the two devices. The IR LEDs composing the marker are first segmented from the microscope images. By knowing their corresponding three‐dimensional (3D) position in the optical tracker reference system, it is possible to find the transformation matrix between the referential of the two devices. Registration and calibration parameters can be extracted directly from that transformation. In addition, since the zoom and focus can be modified by the surgeon during the operation, we propose a spline based method to update the camera model to the new setup. Results The proposed technique is currently being used in an augmented reality system for image‐guided surgery in the fields of ear, nose and throat (ENT) and craniomaxillofacial surgeries. Conclusions The results have proved to be accurate and the technique is a fast, dynamic and reliable way to calibrate and register the two devices in an OR environment. Copyright © 2008 John Wiley & Sons, Ltd.