Axonal transport of neurofilaments (NFs) has long been considered to be regulated by phosphorylation, although recent studies have challenged this hypothesis. Our prior analyses of axonal transport in optic axons demonstrated two distinct NF transport rates that spatially and temporally correlated with changes in NF phosphorylation. In our prior studies, we focused on subunits already within axons. Re-examination of these data using additional approaches and examining additional earlier time points have allowed us to calculate rates at which subunits transport out of retinas and into optic axons. NF subunits were radiolabeled by intravitreal injection of 35S-methionine. NF axonal transport was monitored by following the location of the front of radiolabeled subunits immunoprecipitated from retinas and segments of optic axons, which demonstrated four distinct transport rates. Subunits within retinas exhibited the fastest rate, and underwent a 50% slowing upon exiting the retina and entering optic axons. While this slowing could be due to a regional caliber increase and/or regional increase in NF phosphorylation within the first segment, prior studies indicated that inhibition of phosphatase activities increased NF phosphorylation within retinas and slowed NF subunit exit from retinas to a degree similar to that normally observed within the first segment of axons, suggesting that regional phosphorylation played a major role in slowing of NF transport following their exit from the retina. These findings provide additional support for the notion that phosphorylation regulates NF axonal transport.