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Purpose: To prospectively define the setup error and the interfraction prostate localization accuracy of the planning target volume (PTV) in the presence of an endorectal balloon (ERB) device. Patients and Methods: Weekly portal images (PIs) of 15 patients undergoing external-beam radiotherapy were analyzed. Displacements of the isocenter and the center of the ERB were measured. The setup and target motion variability were assessed with regard to the position variability of the ERB. Results: The setup error was random and target motion variability was largest in the craniocaudal direction. The mean displacement of the isocenter was 2.1 mm (+/-1.2 mm SD [standard deviation]), 2.4 mm (+/-2.2 mm SD), and 3.8 mm (+/-4.0 mm SD) in the left-right, craniocaudal, and anteroposterior directions, respectively (p=0.1). The mean displacement of the ERB was 2.0 mm (+/-1.4 mm SD), 4.1 mm (+/-2.0 mm SD), and 3.8 mm (+/-3.3 mm SD; p=0.03). Setup margin and internal margin contributed equally to the PTV margin. Cumulative placement insecurity of the field and the ERB together was 4.0 mm (+/-2.1 mm SD) laterally, 6.4 mm (+/-2.5 mm SD) craniocaudally, and 7.7 mm (+/-7.0 mm SD) anteroposteriorly. The 95% CIs (confidence intervals) were 2.9-5.2 mm, 5.1-7.8 mm, and 3.8-11.5 mm. In 35% of cases, the estimation of the dorsal margin exceeded 1 cm. Conclusion: Margin estimate dorsally may exceed 1 cm and on-line position verification with an ERB cannot be recommended for dose escalation>70 Gy. [ABSTRACT FROM AUTHOR]