A method of calibrating a positioning system in a radiation therapy system is disclosed, including a radiation therapy unit having a fixed radiation focus. The method includes the steps of irradiating a calibration tool including at least one reference object, capturing at least one two-dimensional image including cross-sectional representations of reference objects of the calibration tool and determining image coordinates of the representation of each reference object. Based on the reference objects image coordinates, positions of the reference objects in the stereotactic coordinate system relative to an origin of the calibration tool and the position of the origin of the calibration tool relative to the imaging unit, a position difference between the position of the calibration tool in the stereotactic coordinate system and a position of the calibration tool in an imaging system coordinate system including a translational and rotational position difference is calculated.
