Abstract Myelinated axons, which had been prepared from bovine brain white matter employing the flotation method, were extracted with Triton X-100 in a low ionic strength buffer containing 4 mM Mg 2+. After delipidation of the detergent-resistant, residual material with chloroform-methanol, glial fibrillary acidic protein (GFAP) was solubilized with 6 M urea. It was enriched by ion exchange chromatography in the presence of 6 M urea on CM-Sepharose CL-6B at pH 5 and on DE52-cellulose at pH 7.6, respectively. The final purification was achieved by affinity chromatography on single-stranded DNA-cellulose in 6 M urea. Employing SDS-polyacrylamide gel electrophoresis, the molecular weight of the purified GFAP was determined to be 51,000. 2D-polyacrylamide gel electrophoresis revealed a major protein constituent of pI 4.7 to 4.8, accompanied by 3 acidic isoelectric variants. Upon incubation at 37° C in the presence of 150 mM KCl, GFAP assembled into 10 nm-filaments.