To compare the accuracy of imaging modalities, immobilization, localization, and positioning techniques in patients with prostate cancer. Thirty-five patients with prostate cancer had gold marker seeds implanted transrectally and were treated with fractionated radiotherapy. Twenty of the 35 patients had limited immobilization; the remaining had a vacuum-based immobilization. Patient positioning consisted of alignment with lasers to skin marks, ultrasound or kilovoltage X-ray imaging, optical guidance using infrared reflectors, and megavoltage electronic portal imaging (EPI). The variance of each positioning technique was compared to the patient position determined from the pretreatment EPI. With limited immobilization, the average difference between the skin marks' laser position and EPI pretreatment position is 9.1 +/- 5.3 mm, the average difference between the skin marks' infrared position and EPI pretreatment position is 11.8 +/- 7.2 mm, the average difference between the ultrasound position and EPI pretreatment position is 7.0 +/- 4.6 mm, the average difference between kV imaging and EPI pretreatment position is 3.5 +/- 3.1 mm, and the average intrafraction movement during treatment is 3.4 +/- 2.7 mm. For the patients with the vacuum-style immobilization, the average difference between the skin marks' laser position and EPI pretreatment position is 10.7 +/- 4.6 mm, the average difference between kV imaging and EPI pretreatment position is 1.9 +/- 1.5 mm, and the average intrafraction movement during treatment is 2.1 +/- 1.5 mm. Compared with use of skin marks, ultrasound imaging for positioning provides an increased degree of agreement to EPI-based positioning, though not as favorable as kV imaging fiducial seeds. Intrafraction movement during treatment decreases with improved immobilization.