The differentiation of glia in the central nervous system is not well understood. A major problem is the absence of an objective identification system for involved cells, particularly the early-appearing radial glia. The intermediate filament structural proteins vimentin and glial fibrillary acidic protein have been used to define the early and late stages, respectively, of astrocyte development. However, because of the non-specificity of vimentin and the temporal overlap in expression patterns of both proteins, it is difficult to refine our view of the process. This is especially true of the early differentiation events involving radial glia. Using the developmentally-expressed intermediate filament-associated protein IFAP-70/280 kD in conjunction with vimentin and glial fibrillary acidic protein markers, a comprehensive investigation of this problem was undertaken using immunofluorescence microscopy of developing rat spinal cord (E13-P28 plus adult). The phenotypes of the cells were defined on the basis of their immunologic composition with respect to IFAP-70/280 kD (I), vimentin (V) and GFAP (G). A definitive immunotype for radial glia was established, viz, I+/V+/G-; thus reliance upon strictly morphological criteria for this early developmental cell was no longer necessary. Based upon the immunotypes of the cells involved, four major stages of macroglial development were delineated: (1) radial glia (I+/V+/G-); (2) macroglial progenitors (I+/V+/G+); (3) immature macroglia (I-/V+/G+); and (4) mature astrocytes (I-/V+/G+ primarily in white matter and I-/V-/G+, the predominant type in gray matter). It is of interest to note that the cells of the floor plate were distinguished from radial glia by their lack of IFAP-70/280 kD immunoreactivity. Introduction of the IFAP-70/280 kD marker has therefore provided a more refined interpretation of the various differentiation stages from radial glia to mature astrocytes.