OBJECTIVE: To assess intra- and inter-site soft tissue variability by dual energy X-ray absorptiometry (DXA). DESIGN: Cross-sectional trial. SETTING: Three medical research institutions. SUBJECTS: Five humans (in vivo) and four phantoms (in vitro), configured from two whole body phantoms with artificial skeletons and thickness overlays. INTERVENTIONS: Duplicate total-body DXA scans were performed on all subjects at each institution within a 15 d period. RESULTS: All intra-site coefficients of variation (CV) were < 0.5% for total tissue mass, but in vitro and in vivo Cvs were 7.2% and 2.3% for fat mass (FM) and 2.5% and 0.9% for lean mass (LM), respectively. Several total-body and regional FM and LM measurements were significantly different between sites (P < 0.05), with percent differences between sites ranging from 2.6-13.3% for FM and from 1.6-13.6% for LM. Site 2 was consistently lower for FM and Site 3 was consistently lower for LM. CONCLUSIONS: These results stress the need for both rigorous and standardized cross-calibration procedures for soft tissue measurement by DXA.