Magnetic resonance imaging (MRI) and computerized tomography (CT) are promising reference methods for quantifying whole body and regional skeletal muscle mass. Earlier MRI and CT validation studies used data-acquisition techniques and data-analysis procedures now outdated, evaluated anatomic rather than adipose tissue-free skeletal muscle (ATFSM), studied only the relatively large thigh, or found unduly large estimation errors. The aim of the present study was to compare arm and leg ATFSM cross-sectional area estimates (cm2) by using standard MRI and CT acquisition and image-analysis methods with corresponding cadaver estimates. A second objective was to validate MRI and CT measurements of adipose tissue embedded within muscle (interstitial adipose tissue) and surrounding muscle (subcutaneous adipose tissue). ATFSM area (n = 119) by MRI [38.9 +/- 22.3 (SD) cm2], CT (39.7 +/- 22.8 cm2), and cadaver (39.5 +/- 23.0 cm2) were not different (P > 0.001), and both MRI and CT estimates of ATFSM were highly correlated with corresponding cadaver values [MRI: r = 0.99, SE of estimate (SEE) 3.9 cm2, P < 0.001; and CT: r = 0.99, SEE = 3.8 cm2, P < 0.001]. Similarly good results were observed between MRI- and CT-measured vs. cadaver-measured interstitial and subcutaneous adipose tissue. For MRI-ATFSM the intraobserver correlation for duplicate measurements in vivo was 0. 99 [SEE = 8.7 cm2 (2.9%), P < 0.001]. These findings strongly support the use of MRI and CT as reference methods for appendicular skeletal muscle, interstitial and subcutaneous adipose tissue measurement in vivo.