The central nervous system (CNS) contains several types of neuroglial cells. In the present study, we characterized different types of glial cells in rat CNS by using single and combined immuno- and enzyme-histochemical methods, and immunofluorescence techniques. Two recently developed monoclonal antibodies (mAbs) against rat macrophages-associated antigens appeared to recognize a subpopulation of glial cells in the CNS of normal adult rats. These ED4- and ED8-positive glial cells were predominantly located in the white matter of adult rat CNS and shared morphological features with microglia. ED4 and ED8 were applied in a double staining combined with mAbs and an antiserum raised against galactocerebroside (GalC) to identify oligodendrocytes, or with anti-glial fibrillary acidic protein antiserum (GFA) to identify astrocytes. We also used a mAb against myelin basic protein (MBP) to identify oligodendrocytes. It appeared that ED4 and ED8 recognized a subpopulation of oligodendrocytes. MAbs against GalC and MBP recognized cells in an immunoperoxidase staining with a morphology identical to that of the ED8-positive cells and part of the ED4-positive cells. Frozen sections of Lewis rats CNS with acute experimental allergic encephalomyelitis (EAE) were investigated, where infiltrating brain macrophages could be found which stained positively with ED4 and ED8 as well as with the monocyte/macrophage mAbs ED1 and ED2. These brain macrophages did not stain when GalC, MBP and GFA markers were applied. Furthermore, ED4+GalC+ and ED8+GalC+ oligodendrocytes were present in the CNS white matter of EAE animals with similar appearance as in normal adult rats. With the currently used markers, we could not detect a third type of neuroglial cell, besides the astrocytes and oligodendrocytes. Thus, none of our anti-macrophage monoclonals recognized the presumptive microglia. Only under pathological conditions, e.g., in inflammatory infiltrates in the course of EAE, could brain macrophages be detected in the CNS parenchyma and only in the direct vicinity of blood vessels, indicating their hematogenous origin.