Quantitative ultrasound (QUS) assessment of bone may permit an assessment of bone properties currently not available by bone densitometry techniques. To explore the effects of the quantity of bone mineral on acoustic parameters, we carried out an in vitro study of the impact of demineralization on attenuation of ultrasound in trabecular bone. Ten fresh cubes of trabecular bone obtained from bovine distal femurs were progressively demineralized using formic acid solution. The progression of demineralization was controlled by monitoring the specimen bone mineral density (BMD) using dual x-ray absorptiometry (DXA). At five stages of demineralization-0% (baseline), 25%, 50%, 75%, and 100% (all mineral removed)-the US properties of the specimens were assessed (Walker Sonix UBA 575+). The US parameters investigated were broadband ultrasound attenuation (BUA) and ultrasound attenuation in bone (UAB). Both DXA and QUS measurements were made along the three orthogonal axes of each cube. Our results demonstrated significant variability in both BUA and UAB along the three principle axes of the cubes whereas BMD did not differ in the different directions. A strong but nonlinear correlation was found between BMD and US attenuation. A reduction in BMD to 50% of the baseline values resulted in BUA (UAB) reduction to 25% and 19%, respectively. A random effect model analysis supported a multiplicative relationship between BMD and the US parameters. US attenuation is a sensitive indicator of bone mineral changes with nonlinear dependence on bone mineral loss. Bone collagen structure reinforced by hydroxyapatite crystal accounts for fundamental US characteristics. Ultrasound attenuation associated with trabecular orientation is basically dominated by the mineral spread in a collagen framework.