Dual-energy x-ray absorptiometry (DXA) is widely applied for estimating body fat. The percentage of body mass as fat (%fat) is predicted from a DXA-estimated R(ST) value defined as the ratio of soft tissue attenuation at two photon energies (e.g., 40 keV and 70 keV). Theoretically, the R(ST) concept depends on the mass of each major element in the human body. The DXA R(ST) values, however, have never been fully evaluated by measured human elemental composition. The present investigation evaluated the DXA R(ST) value by the total body mass of 11 major elements and the DXA %fat by the five-component (5C) model, respectively. Six elements (i.e. C, N, Na, P, Cl and Ca) were measured by in vivo neutron activation analysis, and potassium (i.e. K) by whole-body (40)K counting in 27 healthy adults. Models were developed for predicting the total body mass of four additional elements (i.e. H, O, Mg and S). The elemental content of soft tissue, after correction for bone mineral elements, was used to predict the R(ST) values. The DXA R(ST) values were strongly associated with the R(ST) values predicted from elemental content (r = 0.976, P < 0.001), although there was a tendency for the elemental-predicted R(ST) to systematically exceed the DXA-measured R(ST) (mean +/- SD, 1.389 +/- 0.024 versus 1.341 +/- 0.024). DXA-estimated %fat was strongly associated with 5C %fat (24.4 +/- 12.0% versus 24.9 +/- 11.1%, r = 0.983, P < 0.001). DXA R(ST) is evaluated by in vivo elemental composition, and the present study supports the underlying physical concept and accuracy of the DXA method for estimating %fat.