Axonal neurofilaments are composed of light (NF-L), medium (NF-M), and heavy (NF-H) subunits which are sequentially expressed during axonal development. In retina, NF-L and NF-M appear prenatally, followed postnatally by NF-H which occurs at about the time axons are reaching their target tissue. Phosphorylation of the NF-H protein occurs after it has first appeared in the axon. Phosphorylated NF-H is also downregulated in regenerating peripheral nerves. These observations lead to the hypothesis that NF-H stabilizes axons, thereby inhibiting their ability to grow. We have previously shown that adult mouse retinal ganglion cells (RGCs) can extend neurites in vitro and that these neurites reexpress the developmental protein GAP-43. Here we ask whether the expression of neurofilament proteins in the growing adult RGC axons involves the recapitulation of development. Adult mice which had a priming lesion of the optic nerve and Embryonic Day 15 mouse retinas were explanted onto laminin substrates and grown in culture. After 2-4 days the growing neurites were stained with a battery of monoclonal antibodies against differentially phosphorylated versions of the neurofilament subunits. Both adult and embryonic neurites were highly immunoreactive for NF-L and NF-M. Only the adult neurites stained with antibodies against phosphorylated NF-H. There was no immunoreactivity in the embryonic explants. This indicates that regrowing adult RGC axons maintain their adult cytoskeletal properties and can nevertheless regenerate.