Ser55 within the head domain of neurofilament light chain (NF-L) is transiently phosphorylated by protein kinase A, and phosphorylation of this residue is thought to regulate assembly of neurofilaments. To understand how Ser55 phosphorylation influences NF-L assembly, wild-type and mutant NF-L genes in which Ser55 was mutated to alanine, so as to prevent phosphorylation, or to aspartate, so as to mimic permanent phosphorylation, were transfected into mammalian cells that contain or do not contain an endogenous intermediate filament network. Wild-type and mutant NF-Ls localised to the Triton X-100-insoluble fraction, which suggests that phosphorylation of Ser55 does not inhibit assembly of NF-L and NF-L/ vimentin polymers at or below the tetrameric stage. Immunofluorescence microscopy of transfected cells demonstrated that the wild-type and mutant NF-Ls all colocalised with vimentin to produce similar filamentous arrays. However, in cells lacking an endogenous intermediate filament network, the aspartate mutant produced a pattern of staining different from that of the wild-type or alanine mutant. These results suggest that phosphorylation of NF-L Ser55 is not a mechanism that precludes assembly of neurofilaments from monomers into intermediate filament structures but that phosphorylation/dephosphorylation of this residue might confer more subtle characteristics on neurofilament assembly properties and architecture.