We have found recently that white matter astrocytes in the spinal cord constitutively express immunoreactivity for Mts1 (S100A4) protein and that this expression is up‐regulated ipsilaterally after sciatic nerve or dorsal root injury. Here, we have studied the expression pattern of Mts1 throughout the rat central nervous system (CNS). We found Mts1 immunoreactivity in myelinated tracts such as the olfactory tract, optic nerve, corpus callosum, internal capsule, fimbria, and spinal cord funiculi but not in cerebellar white matter. Mts1‐immunoreactive (IR) cells were consistently astrocytic (glial fibrillary acidic protein positive). In addition to myelinated tracts, Mts1 immunoreactivity was also present in a few nonmyelinated or poorly myelinated areas, such as pituitary gland, olfactory bulb, and around the lateral ventricle. Based on location, three Mts1‐IR astrocyte groups were distinguished: 1) astrocytes at the surfaces of the CNS, i.e., adjacent to the cerebrospinal fluid, organized perpendicularly to the bundles of axonal tracts; 2) astrocytes located in parallel to, and inserted between, axonal bundles; and 3) clusters of astrocytes around the lateral ventricle and in the olfactory bulb. We further analyzed the relationship between Mts1 immunoreactivity and the development of CNS fiber tracts by combining staining for Mts1 and myelin basic protein (MBP). Mts1 immunoreactivity appeared postnatally in recently myelinated areas. During the development of corpus callosum and the optic tract, Mts1 immunoreactivity was concentrated at the frontier of myelination. The developmental expression pattern suggests a role of Mts1‐IR astrocytes in the maturation of myelinated fiber tracts. The preferential localization of Mts1 to the subpial region in the mature CNS suggests that Mts1 participates in astrocyte‐mediated CNS‐cerebrospinal fluid exchange. J. Comp. Neurol. 424:269–282, 2000. © 2000 Wiley‐Liss, Inc.