An MRI technique with high selectivity and sensitivity to the signal components in the chemical shift range of methylene and methyl protons of fatty acids has been developed for noninvasive assessment of muscular fat in vivo. A spoiled gradient-echo sequence with spatial-spectral excitation by six equidistant pulses with 2°-(−9°)-17°-(−17°)-9°-(−2°) and a multi-echo train (TE = 16, 36, 56, 76, 96, and 116 ms) allowed a series of images to be recorded with a receiver bandwidth of 78 Hz per pixel. SIs from phantoms with lipid contents between 0.1% and 100% were compared to those from pure water. Thirty healthy volunteers underwent fat-selective imaging of their lower leg, and parallel localized proton spectroscopy of the tibialis anterior and the soleus muscle by a single-voxel stimulated echo acquisition mode (STEAM) technique (TR = 2 s, TE = 10 ms, TM = 15 ms). Results show a high correlation (r = 0.91) between fat imaging and the spectroscopic approach in the soleus muscle, considering the percentage total fat content of musculature. The correlation coefficient was clearly lower (r = 0.55) in the tibialis anterior muscle due to signal contaminations from adjacent subcutaneous fat in the images, inhomogeneous fat distribution, and generally lower lipid content in this muscle. Applications of the new imaging technique showed marked intra- and interindividual variability in the spatial distribution of lipids in the musculature of the lower leg. No significant correlation of the muscular fat with the thickness of the subcutaneous fat layer was found. In addition, the body mass index does not appear to determine muscular fat content, except in very obese cases. Magn Reson Med 47:720–727, 2002. © 2002 Wiley-Liss, Inc.