This paper is a step in investigating whether three-dimensional (3D) ultrasound can be used intraoperatively to replace Computed Tomography (CT) for localization of brachytherapy seeds. In order to quantify the accuracy and variability of seed localization without introducing effects due to tissues, we first report our results with test phantoms. An inter- and intra-observer study was performed to assess the variability of 2 3D ultrasound scan acquisition methods: Tilt 3D scanning and pull-back 3D scanning. Seven observers measured the positions of gold seed markers in an agar phantom twice in each of the three orthogonal image planes. An analysis of variance (ANOVA) was performed to determine the intra- and inter-observer standard errors of measurement (SEM) and the minimum detectable changes in marker position (deltap). Average intra- and inter-observer SEMs for the tilt scan 3D image were 0.36 and 0.40 mm, respectively. Measurements of the pull-back scan 3D image yielded average intra- and inter-observer SEM of 0.46 and 0.49 mm, respectively. A paired difference analysis showed that the lower SEM for the tilt 3D scan image were statistically significant at a significance level of alpha= 0.05. The accuracy of the US measurements was tested by determining marker coordinates from CT images of the phantom in a stereotactic head frame. CT coordinates were matched to the ultrasound (US) coordinates by means of an affine transform. Average matching errors in x, y, and z were 0.02, 0.10, and -0.02 mm, respectively.