Previous bone comparison studies between subjects with and without Down syndrome (DS) were performed using bone mineral density (BMD) as the dependent variable, and mainly focused on lumbar spine region. The purpose of this study was to compare bone mineral mass adjusted for bone and body size, in limbs, lumbar spine, and femoral neck between males and females with and without DS. Subjects were 66 females (33 with DS) and 68 males (34 with DS) aged 14-40 years. Analysis of covariance (ANCOVA) was used to analyze the main and interaction effects of gender and condition on bone mineral mass. For this purpose, adjusted bone mineral content (BMC) (for bone area, height, and age), volumetric bone mineral density (vBMD) (for age), and composite indices of femoral neck strength (for age), were used as the dependent variables, corrected additionally for body composition variables selected by regression analysis. ANCOVA revealed lower lumbar spine vBMD in DS than in control subjects with (-5%, P = 0.013), or without body weight adjustments (-6%, P = 0.003). In femoral neck, the mean of each strength measure was also lower in DS than in control subjects. Mean differences between groups were, with and without additional adjustments for fat mass, respectively, -8% (P = 0.009), and -13% (P < 0.001) for compressive strength, -11% (P = 0.036), and -16% (P = 0.004) for bending strength, and -7% (P = 0.031), and -11% (P = 0.002) for impact strength. These lumbar spine and femoral neck differences between groups were highest in young adults (> 20 years) and not significant in adolescents. No interaction effect was observed between gender and condition. In conclusion, DS was shown to be a risk factor for low vBMD in lumbar spine, and for diminished bone strength relative to the loads that the femoral neck must bear. Body composition did not reach statistical significance as predictor of bone differences in these sites between subjects with and without DS, suggesting that other factors may be involved in this detrimental bone status, particularly in young adults compared with adolescents.