The appearance of reactive astrocytes following brain injury was investigated in 4-week-old rats with special reference to their proliferation and chronological changes in the cytoskeletal proteins. Two days after the injury, glial fibrillary acidic protein (GFAP)-positive cells had increased in number around the lesion and spread to the entire ipsilateral cortex by 3 days after the injury. To investigate the distribution of mitotic cells and its chronological change, immunohistochemical staining with monoclonal antibody to bromodeoxyuridine (BrdU) was performed. BrdU-positive cells began to appear around the lesion and spread to the entire ipsilateral cortex by 3 days and their distribution was the same as that of GFAP-positive cells. To investigate the association of GFAP-positive cells with cell division, double labeling experiments using [3H]thymidine autoradiography and immunohistochemical staining with antiserum to GFAP were performed. Cells doubly labeled with GFAP and [3H]thymidine were localized in the area adjacent to the lesion, in the molecular layer of the cortex and in the white matter. By contrast, none of the cells were doubly labeled in the IInd to VIth layers of the cortex. Furthermore, only astrocytes in the former areas expressed vimentin transiently from 2 to 10 days after the injury. In the rats administered vincristine, cells arrested during mitosis were found in the regions which express vimentin. From these results, it was suggested that astrocytes in the molecular layer of the cortex and the white matter adjacent to the lesion proliferated in response to the injury and expressed vimentin transiently, then acquired GFAP, and that astrocytes in the IInd to VIth layers of the cortex became reactive astrocytes without mitosis.