PURPOSE: To quantify the interfraction variability in prostate and seminal vesicle (SV) positions during a course of intensity-modulated radiotherapy (IMRT) using an integrated computed tomography (CT)-linear accelerator system and to assess the impact of rectal and bladder volume changes. METHODS AND MATERIALS: We studied 15 patients who had undergone IMRT for prostate carcinoma. Patients had one pretreatment planning CT scan followed by three in-room CT scans per week using a CT-on-rails system. The prostate, bladder, rectum, and pelvic bony anatomy were contoured in 369 CT scans. Using the planning CT scan as a reference, the volumetric and positional changes were analyzed in the subsequent CT scans. RESULTS: For all 15 patients, the mean systematic internal prostate and SV variation was 0.1 +/- 4.1 mm and 1.2 +/- 7.3 mm in the anteroposterior axis, -0.5 +/- 2.9 mm and -0.7 +/- 4.5 mm in the superoinferior axis, and 0.2 +/- 0.9 mm and -0.9 +/- 1.9 mm in the lateral axis, respectively. The mean magnitude of the three-dimensional displacement vector was 4.6 +/- 3.5 mm for the prostate and 7.6 +/- 4.7 mm for the SVs. The rectal and bladder volume changes during treatment correlated with the anterior and superior displacement of the prostate and SVs. CONCLUSION: The dominant prostate and SV variations occurred in the anteroposterior and superoinferior directions. The systematic prostate and SV variation between the treatment planning CT and daily therapy as a result of the rectal and bladder volume changes emphasizes the need for daily directed target localization and/or immobilization techniques.