In mammals neurofilaments (NF) are formed by coassembly of three subunits: NFL, NFM, and NFH (light, medium, and heavy). It had been believed that lampreys have only one subunit, NF180. However, a previous study showed that NF180 could not self-assemble but could coassemble with rat NFL, suggesting the existence of additional NF subunits in lamprey. More recently, we cloned three additional NF subunits. These new subunits and NF180 have now been transfected in combinations into SW13cl.2Vim− cells, which lack endogenous cytoplasmic intermediate filaments. None of the subunits could self-assemble. No combination of NF subunits could form filaments in the absence of lamprey NFL (L-NFL). Assembly occurred at 28°C, but not at 37°C. L-NFL could form thick NF bundles with NF180 but not with NF132 and NF95, which formed only fine filamentous arrays. To determine which parts of the NF subunits are required for filament or bundle formation, we constructed deletion mutants of NF180 and cotransfected them with L-NFL. As with mammalian NF, only constructs with intact head and core domains could form filaments with L-NFL. However, the full length of NF180 was required to form NF bundles. As with NF180, in situ hybridization indicated that mRNA for L-NFL and NF132 was downregulated in identified reticulospinal neurons by 5 weeks after spinal cord transection, but was reexpressed at 10 weeks selectively in those neurons whose axons have a high probability of regenerating. This is consistent with a possible role of NFs in the mechanism of axon regeneration. J. Comp. Neurol. 519:3657–3671, 2011. © 2011 Wiley-Liss, Inc.