Ultrasound (US) measurements of the calcaneus are usually carried out in a region of interest (ROI) at a fixed site relative to a footplate. Recently, US transmission systems have been developed with imaging capability that enable selection of the position of ROI; the region of measurement is always the area of minimum attenuation in the posterior part of the calcaneus. This study compares measurements of broadband ultrasound attenuation (BUA) and speed of sound (SOS) at the variable ROI of minimum attenuation (ROIv) and at fixed coordinates (ROIf). Ultrasound variables were estimated at ROIv and ROIf in 212 female subjects, including 26 patients with osteoporotic fractures. Among the 186 women without fractures, 63 were classified as having osteoporosis on the basis of their vertebral bone density. Precision of BUA and SOS were better at ROIv than at ROIf. BUA was more highly correlated with bone mineral density (BMD) at the lumbar spine and femoral neck at ROIv than ROIf (r = 0.64 for lumbar spine and 0.68 for femoral neck at ROIv versus 0.50 for lumbar spine and 0.54 for femoral neck at ROIf, P < 0.05 for both comparisons). There were no significant differences between the correlations of SOS with axial BMD at ROIv compared with ROIf. Significant difference was found between the areas under the ROC curve for each ultrasound variable at ROIv and ROIf for both groups of patients, subjects with osteoporosis (area under curve = 0.87 for BUA at ROIv versus 0.82 at ROIf, P < 0.05; area under curve = 0.85 for SOS at ROIv versus 0.81 at ROIf, P < 0. 05), and women with fractures (area under curve = 0.93 for BUA at ROIv versus 0.86 at ROIf, P < 0.05; area under curve = 0.91 for SOS at ROIv versus 0.82 at ROIf, P < 0.05). Ultrasound variables measured at ROIv enable improved reproducibility and significantly better differentiation of diseased subjects from healthy individuals as compared with measurements at ROIf.