The purpose of this paper is to study whether the deep freezing of bone and later thawing affects the bone mineral density (BMD) measurement. We used 56 calcanei from 28 adult female New Zealand white rabbits and 102 human calcanei from 51 donors post-mortem (27 men and 24 women, age 30-89). Dual energy x-ray absorptiometry evaluated BMD of the frozen specimen and of the thawed specimen. A main analysis compared BMD of each specimen in the frozen and thawed states. The mean BMD of 224 areas of frozen rabbits' calcanei was 0.31 +/- 0.08 g cm(-2) (95% confidence interval (CI) from 0.30 to 0.32 g cm(-2)) while the mean BMD of thawed rabbits' calcanei was 0.31 +/- 0.08 g cm(-2) (95% CI from 0.30 to 0.32 g cm(-2); paired t-test p > 0.01). The mean BMD of 306 areas of frozen human calcaneus was 0.73 +/- 0.22 g cm(-2) (95% CI from 0.70 to 0.76 g cm(-2)) while the mean BMD of thawed human calcaneus was 0.73 +/- 0.22 g cm(-2) (95% CI from 0.70 to 0.76 g cm(-2); p > 0.01). For both the rabbit and the human calcanei, a Bland-Altman analysis showed a mean difference between the BMD in the frozen and thawed states of 0.00 (limits of agreement, rabbit: -0.07 to 0.08 g cm(-2), human: -0.16 to 0.21 g cm(-2)). A high correlation was observed between calcaneus BMD in the frozen and thawed states (r = 0.94, 0.97, 0.92 and 0.99 respectively in all rabbit calcanei, all human calcanei, immobilized rabbit calcanei and osteopenic human calcanei, all p < 0.01). Bone mineral density is not affected by deep freezing and later thawing of the specimen. Therefore, the specimens need not be thawed to obtain valid and precise BMD measurement. These results are relevant to general musculoskeletal as well as osteoporosis research where the specimens undergo multiple tests in series.