Quantitative ultrasound (QUS) techniques have recently been introduced as alternative methods free of ionizing radiation for non-invasive assessment of skeletal status in osteoporosis. We carried out an in vitro study on bone specimens to investigate whether broadband ultrasound attenuation (BUA) signals are associated with bone structure, specifically with the orientation of the trabeculae, and whether this association is independent of the association between orientation and bone mineral density (BMD) as measured by dual-energy X-ray absorptiometry (DXA). BUA and BMD of 10 cubical specimens of purely trabecular bovine bone were examined along the three principal axes. The relative orientation of the trabeculae with respect to the direction of the ultrasound beam was evaluated on high-resolution conventional radiographs employing a semiquantitative ALIGNMENT score ranging from -2 (for perpendicular) to +2 (parallel). BUA variability was 27.6 dB/MHz, reflecting both inter-specimen (18.2 dB/MHz) and intra-specimen (19.4 dB/MHz) variability at comparable levels and to a much lesser extent reproducibility errors (1 dB/MHz). BUA was 44%-54% larger along the axis of the compressive trabeculae as compared with the two perpendicular axes. BMD and ALIGNMENT showed independent significant associations with BUA. A change in ALIGNMENT from perpendicular to parallel corresponded to a difference in BUA of 36.1 dB/MHz. The substantial level of intra-specimen variability suggests that BUA reflects anisotropical characteristics of trabecular bone. The association of BUA and ALIGNMENT indicates that BUA signals depend of trabecular orientation. This association is independent of BMD, indicating that BUA has considerable potential for non-invasive assessment of bone structure and strength, free of ionizing radiation, and for complementing existing bone densitometry examinations.