Although a strong relationship exists between areal bone mineral density (aBMD) derived from dual-energy X-ray absorptiometry (DXA) and bone strength, the predictive validity of aBMD for osteoporotic vertebral fractures remains suboptimal. The diagnostic sensitivity of DXA may be improved by assessing aBMD within vertebral subregions, rather than relying on an estimate derived from the total area of the vertebra. The objective of this study was to validate a method of measuring subregional vertebral aBMD in vitro using lateral-projection DXA against subregional volumetric BMD (vBMD) measured with peripheral quantitative computed tomography (pQCT). A mixed set of 49 lumbar and thoracic vertebrae from 25 donors were scanned using lateral-projection DXA and pQCT. aBMD and apparent vBMD were measured in 7 vertebral regions (1 total area and 6 subregions) from the lateral DXA scan. vBMD was calculated in anatomically equivalent regions from pQCT scan data, using a customised software program designed to increase efficiency of the analysis process. Significant differences in densitometric parameters between subregions were observed by DXA and pQCT (P < 0.01). Subregional vBMD derived from pQCT was explained by a significant proportion of the variance in DXA-derived aBMD (R 2 = 0.51-0.67, P < 0.05) and apparent vBMD (R 2 = 0.64-0.75, P < 0.05). These results confirm the validity of measuring aBMD in vertebral subregions using lateral-projection DXA. The clinical significance should now be explored.