Margins are used in radiotherapy to assist in the calculation of planning target volumes. These margins can be determined by analysing the geometric uncertainties inherent to the radiotherapy planning and delivery process. An important part of this process is the study of electronic portal images collected throughout the course of treatment. Set-up uncertainties were determined for prostate radiotherapy treatments at our previous site and the new purpose-built centre, with margins determined using a number of different methods. In addition, the potential effect of reducing the action level from 5 mm to 3 mm for changing a patient set-up, based on off-line bony anatomy-based portal image analysis, was studied. Margins generated using different methodologies were comparable. It was found that set-up errors were reduced following relocation to the new centre. Although a significant increase in the number of corrections to a patient's set-up was predicted if the action level was reduced from 5 mm to 3 mm, minimal reduction in patient set-up uncertainties would be seen as a consequence. Prescriptive geometric uncertainty analysis not only supports calculation and justification of the margins used clinically to generate planning target volumes, but may also best be used to monitor trends in clinical practice or audit changes introduced by new equipment, technology or practice. Simulations on existing data showed that a 3 mm rather than a 5 mm action level during off-line, bony anatomy-based portal imaging would have had a minimal benefit for the patients studied in this work.