Neurofilaments are composed of 3 polypeptides designated NF-H, NF-M, and NF-L, all of which are subject to posttranslational phosphorylation. It has been suggested that phosphorylation of the NF-L polypeptide can influence the assembly of NF-L into filaments, but the sites at which NF-L is phosphorylated are unknown. To locate these phosphorylation sites, we have identified phosphopeptides of NF-L by labeling them with 32P both in vitro and in cultured neurons and also by observing their change in chromatographic behavior after they have been treated with phosphatase. We report here that serine 473, in the carboxy-terminal tail domain of NF-L, is a major substrate in vitro for protein kinases endogenous to a crude cytoskeleton-containing fraction. Moreover, serine 473 is a major phosphorylation site in vivo; in neurofilaments isolated from rat spinal cord, approximately 73% of serine 473 was phosphorylated, and accounted for at least one-third of the total phosphate associated with NF-L. The identification of this phosphorylation site in NF-L provides a criterion for identifying the protein kinase that phosphorylates NF-L and raises the question of its function.