1H MR spectroscopy (MRS) has proved to be a valuable noninvasive tool to measure intramyocellular lipids (IMCL) in research focused on insulin resistance and type II diabetes in both humans and rodents. An important determinant of IMCL is the muscle fiber type, since oxidative type I fibers can contain up to three times more MCL than glycolytic type II muscle fibers. Because these different muscle fiber types are inhomogeneously distributed in rodent muscle, in the present study we investigated the distribution of IMCL within the rat tibialis anterior muscle (TA) in vivo using single-voxel 1H MRS along with the muscle fiber distribution in the TA ex vivo determined from immunohistological assays. IMCL levels in the TA differed by up to a factor of 3 depending on the position of the voxel. The distribution of IMCL over the TA cross section was not random, but emerged in a pattern similar to the distribution of the predominantly oxidative muscle fiber types. Dietary interventions, such as high-fat feeding and 15 hr of fasting, did not significantly change this typical fiber type-dependent pattern of IMCL content. These results stress the importance of voxel positioning when single-voxel 1H MRS is used to study IMCL in rodent muscle. © 2006 Wiley-Liss, Inc.