The aim of the study was to investigate the effect of three different regions of interest (ROIs) varying in size and shape on broadband ultrasound attenuation (BUA) measurements of the calcaneus. Two hundred and sixty-five postmenopausal Caucasian women participated in this study. In 43 women osteoporotic fractures were documented on spinal radiographs. Bone mineral density (BMD) measurements of the lumbar spine and the femur were made using dual-energy X-ray absorptiometry. BUA measurements were obtained at a circular ROI automatically determined by the imaging system (ROIc), at a manually traced irregular ROI encompassing the posterior part of the calcaneus (ROIi), and at an anatomical square ROI located in the posterior part of the calcaneus (ROIs). Reproducibility was better in ROIc than in ROIi and ROIs. High correlations were found between BUA measurements with ROIc and ROIs (r = 0.981, P < 0.0001) as well as between those with ROIc and ROIi (r = 0.965, P < 0.0001). There were no significant differences between the correlations of BUA with axial BMD at ROIc compared with ROIi and ROIs. No significant difference was found between the areas under the ROC curve at ROIi, ROIc, and ROIs for women with fractures. The results show that superior reproducibility makes ROIc the most appropriate region of BUA measurement in a comparison with ROIi and ROIs.