With the availability of in-room CT-guided radiotherapy, positional uncertainties caused by both setup errors and internal organ motion may be minimized. However, it is still unclear what would be the quantitative dosimetric benefits from using such procedure in daily target localization. In this study we will examine the dosimetric impact of daily CT alignment for prostate cancer radiotherapy. Utilizing the CT-on-rails, patients can be scanned on the treatment couch in the treatment position immediately prior to radiotherapy. In an IRB-approved study, fifteen prostate patients were scanned 3 times each week during their 8-week treatments for an average of 24 scans per patient. Three cases were selected for this introductory study, two with small prostate center-of-volume (COV) variations relative to bony reference (Pat01sPat12) and one with large variations (Pat07). For each case two 8-field, 6MV IMRT plans were designed on the simulation CT. The goal was to treat the PTV (prostate + seminal vesicles (SV) + margin) to 75.6Gy while sparing the rectum and bladder. Plan A used clinical margins (5mm posterior + 1cm other directions). Treatment parameters were imported and re-calculated on all subsequent CT scans, with the beam isocenter determined using skin marks (fiducials) to create the 1cm,fiducials trial. Next, Plan A was imported and recalculated a second time, utilizing ultrasound alignment of the prostate (shifts recorded during actual patient treatments) with BAT (NOMOS Corp.) to create the 1cm,BAT trial. This reflects the current practice at our institution. Plan B used a smaller margin based on our initial experience of the daily CT-guided geometric correction procedure (3mm in all directions). Plan B was imported and re-calculated on all subsequent CT scans, with the beam isocenter determined relative to the daily prostate COV to create the 3mm, COV trial. Seventy-three daily CT images were used for the 3 cases and 216 dose distributions were collected over the course of treatment. The 1cm, fiducials and 1cm, BAT plans provided the same average prostate coverage. For two cases with small prostate movement, 75.6Gy covered 96% (Pat01) and 99% (Pat12) of the prostate with the 1cm plans. For the case with above-average movement (Pat07), the average %-volume of prostate ≥75.6Gy fell to 89% with 1cm, fiducials and 1cm, BAT. When the 3mm, COV method was used instead, every case was within 4% of the planned prostate coverage (98% Pat01, 96% Pat07, 100% Pat12). The range of daily variations in the prostate dose also decreased when the 3mm, COV method was utilized. None of the three techniques displayed a consistent advantage in the SV 75.6 Gy-coverage. The average coverage ranged from 85% to 98%, compared to the planned 99% to 100% coverage. The prostate alignment techniques did not account for independent SV motion, and the margins were insufficient to fully encompass that motion. In order to assure SV dose coverage additional margins should be added or an adaptive alignment and correction method should be implemented to account for the daily position of the SV separately from the prostate. The dose-volume histograms show the potential dose reductions to the bladder and rectum offered by the 3mm, COV method compared to both 1cm methods in the planning and the actual treatment deliveries. The actual bladder volume ≥40Gy and ’70Gy was reduced (by 19–22% at 40Gy, 12–17% at 70Gy). The actual rectal wall volume ≥40Gy and ≥70Gy decreased (by 8–17% at 40Gy, 1–10% at 70Gy). The uncertainty in the daily rectal and bladder doses decreases with the 3mm, COV method. By utilizing a daily CT prostate alignment technique, the planning margin of the prostate may be reduced to 3 mm. CT-guided prostate localization also improves the daily delivery accuracy, increasing the average prostate coverage. However the 3mm margin may be too small to ensure SV coverage. Additional margin or alternative alignment strategies should be employed for the daily localization of the SV