The reassembly process of purified native (phosphorylated) and enzymatically dephosphorylated bovineneurofilament (NF) subunits was studied to delineate how NF triplet proteins assemble together into intermediate-sized filaments in vitro. We determined the time course for reassembly, the ultrastructural characteristics of reassembled NFs, and the topographical disposition of NF protein subdomains within reassembled NFs using quantitative biochemical techniques, negative staining and immunoelectron microscopy. Our data indicate that: (1) ≈50% of the purified NF subunit proteins assembled within 30 min from the start of reassembly into 10- to 12-nm filaments, and by 90 min ≈85–90% of the NF proteins were reassembled, (2) low concentrations (0.15–0.5 mg/ml) of purified NF proteins were able to reassemble into long filaments, (3) the rate and ability of native phosphorylated and dephosphorylated NF proteins to assemble into NFs were comparable, (4) negative staining revealed a periodicity of ≈18–22 nm and a protofilamentous substructure in reassembled NFs, (5) immunoelectron microscopy using domain specific anti-NF monoclonal antibodies (mAbs) to all 3 NF proteins demonstrated specific labeling patterns corresponding to the spatial relationships of subdomains within reassembled NFs, and (6) negative staining and immunolabeling revealed that reassembled NFs are very similar to isolated native NFs. We conclude that purified mammalian axonal NF triplet proteins, independent of their phosphorylation state, rapidly and efficiently reassemble in vitro to generate characteristics 10-nm filaments. Furthermore, immunological analysis reveals that the rod domains of NF-H, NF-M and NF-L are buried within the reassembled NF, whereas the head domain of NF-M and the tail domains of all 3 NF proteins remain exposed following reassembly.