Mammalian neurofilaments are composed of three subunit polypeptides with approximate molecular weights of 200 000, 150 000, and 70 000 (P200, P150, and P70). These subunits were separated by ion-exchange chromatography in the presence of 8 M urea. The P200 polypeptide was differentially eluted on a diethylaminoethyl (DEAE) column. The P70 and P150 polypeptides obtained after the DEAE column were separable on a hydroxylapatite column. Under neurofilament assembly conditions, only the P70 polypeptide was able to reassemble into an intermediate filament in the absence of the other two polypeptides. The P150 and P70 polypeptides copolymerized into an intermediate filament, only if P70 was present. These results suggest that the P70 polypeptide forms the core of the filament and the other two polypeptides are tightly associated accessory proteins.