The importance of optimal daily patient positioning has been stressed in order to ensure treatment reproducibility and gain in accuracy and precision. We report our data on the 3D setup uncertainty during radiation therapy for prostate cancer using the CT image fusion technique. Ten consecutive patients scheduled for radiation therapy for prostate cancer underwent 5 prone position CT scans using an individualized immobilization cast. These different setups were analyzed using the image fusion module of the ERGO 3D-Line Medical System (Milan, Italy) treatment planning system. The isocenter and the body marker displacements were measured. The 3D isocenter dislocations were quantified: systematic error was sigma(3D) = 3.9 mm, whereas random error was sigma(3D) = 1 mm. The mean of the minimum displacements was 0.2 +/- 1 mm showing that the immobilization device used allows an accurate setup to be obtained. Single direction errors were also measured showing systematic errors, sigma(AP), = 2.6 mm, sigma(LL) = 0.6 mm, SigmaSI = 3 mm in the anterior-posterior, latero-lateral, superior-inferior direction, respectively. Related random errors were sigma(AP), = 1 mm, sigma(LL) = 0.6 mm, sigma(SI) = 1.2 mm. In terms of accuracy, our uncertainties are similar to those reported in the literature. By applying the CT image fusion technique, a 3D study on setup accuracy was performed. We demonstrated that the use of an individualized immobilization system for prostate treatment is adequate to obtain good setup accuracy, as long as a high-quality positioning control method, such as the stereoscopic X-ray-based positioning system, is used.