This study presents a new method for objective assessment of the distraction regenerate using three-dimensional quantitative computer tomography (3D-QCT). The distraction specimens of 16 sheep that underwent bifocal cranial distraction osteogenesis to reconstruct a critical size defect were used to evaluate this method. To analyse the validity of this method the results were compared to conventional quantitative computer tomography (QCT). Squared correlation coefficients (r(2)) according to Sackett showed a high reliability (r(2) > 70% for the total DO regenerate, DO Regions 2-4). Method comparison according to Bland-Altman (< or =6.25% of measurements out of 95% limits of agreement) showed that three-dimensional computer tomography based bone densitometry is valid for future DO research.