Purpose/Objective: Studies of geometric variability of the prostate have failed to create a concensus on whether to treat external beam radiotherapy (EXRT) patients with a supine or prone setup. More importantly, the dosimetric consequences of this variability have not been quantified. This study is designed to clinically assess the dosimetric differences between supine and prone patient setup for prostate EXRT. Materials/Methods: Twenty patients receiving EXRT for localized prostate adenocarcinoma were prospectively randomized to supine or prone treatment groups. Immobilization was limited to vacuum bags for supine patients and belly boards for prone patients. An initial CT scan was obtained for treatment planning. Beginning at least one week after the start of treatment, ten additional CT scans were obtained on non-consecutive days immediately before or after the daily treatment. For each daily scan, the therapist aligned the patient’s skin marks to the CT lasers in order to mimic the isocentric EXRT setup. To accurately identify the isocenter in the CT scan, metallic markers were placed on these skin marks. A single physician contoured the prostate, seminal vesicles, bladder, and rectum for all scans. The initial treatment plan was then applied to each daily CT scan to determine the variations in the dose volume histograms for these structures. Dose variations were studied for six-field conformal radiotherapy with multiple margin sizes. Results: Preliminary prostate shift analysis was determined from CT contours for ten patients (five supine, five prone, 110 CT scans total). The mean prostate shifts along the three major axes were 1.2mm left (standard deviation: 5.6mm) and 0.2mm left (SD: 4.0mm), 3.1mm posterior (SD: 4.9mm) and 2.4mm posterior (SD: 5.7mm), and 0.4mm cranial (SD: 3.3mm) and 0.9mm caudal (SD: 3.5mm) for supine and prone groups, respectively. These shifts are comparable to those reported from previous studies of prostate motion. The supine and prone shifts are statistically different only in the cranial-caudal direction (p = 0.05). The variances of shifts in all directions for both supine and prone groups were statistically equivalent. Dose variations for prostate, rectum and bladder result from structure movement and size changes. Preliminary dosimetric analysis was based on the same ten patients receiving 75.6 Gy over 42 fractions with six field conformal plans that cover a 1.0cm margin (0.75cm posterior) around the prostate. A two-factor analysis of variance was used with one factor, the patient, nested within the other factor, treatment setup. The analysis indicated that there is no statistically significant difference between supine and prone patient groups for the following dosimetric indicators: mean volume percent of prostate receiving 100% dose, mean volume percent of bladder receiving 80% dose (60.5 Gy), mean absolute volume (cc) of bladder receiving 80% dose, mean volume percent of rectum receiving 80% dose, and mean absolute volume of rectum receiving 80% dose. The lowest p-value was 0.23 for the absolute volume of rectum (7.3 cc supine, 17.2 cc prone). Additional patient data may increase the level of significance for this indicator. Conclusions: Based on preliminary data, there is no statistically significant difference between mean 100% dose coverage of prostate as well as mean 80% dose coverage of bladder and rectum for supine and prone patients. There is also insufficient evidence to indicate a significant difference in prostate position variability for supine and prone patients. There is strong evidence that within-patient variation is greater than within-group variation, suggesting a benefit to obtaining multiple CT scans at the start of treatment in order to estimate this variability.