The quantification of neurofilament light polypeptide (NFL) in biofluids is being clinically used to detect and grade general neuronal damage in neurodegenerative diseases and quantify neuronal injury during acute events like traumatic brain injury. Specific assays that target only particular molecular breakdown products of neurofilaments have the potential to distinguish between various pathologies. Nevertheless, the molecular structure of neurofilament light polypeptide in cerebrospinal fluid remains to be elucidated. We characterized neurofilament light polypeptide in cerebrospinal fluid by size-exclusion chromatography, Western blotting and mass spectrometry. Neurofilament light polypeptide in cerebrospinal fluid was found to be composed of aggregates of fragments of the full-length molecule. These aggregates were sensitive to reduction by dithiothreitol and dissociated to monomeric fragments of 6-12†kDa (Western blot), covering most of the coiled-coil domains of neurofilament light polypeptide. Since only cysteine residues can form disulfide bonds, this points to a role of the single cysteine 322 for maintaining the stability of the aggregates. The sequence region covered by the identified fragments ended just a few amino acids C-terminally of the coiled-coil region at a site which had been previously mapped to a calpain cleavage site in the glial fibrillary acidic protein, which is highly homologous to neurofilament light polypeptide in the coiled-coil region. This cleavage site was also confirmed to be present in bovine neurofilament light polypeptide by in vitro digestion of purified neurofilament light polypeptide with calpain-1. The difference of the molecular weights of the reduced and non-reduced forms of neurofilament light polypeptide suggests that neurofilament light polypeptide in CSF consists of disulfide-linked aggregated fragments, most likely tetramers, or alternately dimers in a complex with another binding partner.