Axonal transport of neurofilament (NFs) is considered to be regulated by phosphorylation. While existing evidence for this hypothesis is compelling, supportive studies have been largely restricted to correlative evidence and/or experimental systems involving mutants. We tested this hypothesis in retinal ganglion cells of normal mice in situ by comparing subunit transport with regional phosphorylation state coupled with inhibition of phosphatases. NF subunits were radiolabeled by intravitreal injection of 35 S-methionine. NF axonal transport was monitored by following the location of the peak of radiolabeled subunits immunoprecipitated from 9 Ο« 1.1 mm segments of optic axons. An abrupt decline transport rate was observed between days 1 and 6, which corresponded to translocation of the peak of radiolabeled subunits from axonal segment 2 into segment 3. Notably, this is far downstream from the only caliber increase of optic axons at 150 Β΅ from the retina. Immunoblot analysis demonstrated a unique threefold increase between segments 2 and 3 in levels of a ''late-appearing'' C-terminal NF-H phospho-epitope (RT97). Intravitreal injection of the phosphatase inhibitor okadaic acid increased RT97 immunoreactivity within retinas and proximal axons, and markedly decreased NF transport rate out of retinas and proximal axons. These findings provide in situ experimental evidence for regulation of NF transport by site-specific phosphorylation.