In the present study, efficient and unbiased stereological techniques to investigate the myelin sheaths of the myelinated fibers in rat white matter were established. In the present design, four tissue blocks were obtained from the entire white matter of rat brain in a uniform, random fashion. Isotropic, uniform random (IUR) sections were ensured by the use of the isector technique. One section with the thickness of 60 nm was cut from the center of each epon block. Eight to 10 fields of vision were randomly photographed under a transmission electron microscope. The total length of the myelinated fibers and the total volume of the myelin sheaths in the white matter were the products of the length density, volume density, and the volume of the white matter obtained with the Cavalieri principle. The mean areas of the myelinated fibers profiles and myelin sheaths were estimated with the point counting technique. The inner and outer perimeters of the myelin sheaths were estimated by the use of a line grid, and the thickness of the myelin sheaths was estimated by direct orthogonal measurements in uniform, random locations. The described methods will provide very useful tools for future quantitative studies of changes in the myelin sheaths of white matter in various experimental conditions and in various neurodegenerative diseases. Anat Rec, 2009. © 2009 Wiley-Liss, Inc.