In the past few years, a number of two-dimensional (2-D) to three-dimensional (3-D) (2-D-3-D) registration algorithms have been introduced. However, these methods have been developed and evaluated for specific applications, and have not been directly compared. Understanding and evaluating their performance is therefore an open and important issue. To address this challenge we introduce a standardized evaluation methodology, which can be used for all types of 2-D-3-D registration methods and for different applications and anatomies. Our evaluation methodology uses the calibrated geometry of a 3-D rotational X-ray (3DRX) imaging system (Philips Medical Systems, Best, The Netherlands) in combination with image-based 3-D-3-D registration for attaining a highly accurate gold standard for 2-D X-ray to 3-D MR/CT/3DRX registration. Furthermore, we propose standardized starting positions and failure criteria to allow future researchers to directly compare their methods. As an illustration, the proposed methodology has been used to evaluate the performance of two 2-D-3-D registration techniques, viz. a gradient-based and an intensity-based method, for images of the spine. The data and gold standard transformations are available on the internet (http://www.isi.uu.nl/Research/Databases/).