The progressive modification of newly synthesized neurofilament proteins (NFPs) during axoplasmic transport in mouse retinal ganglion cell (RGC) neurons was studied after RGC perikarya were pulse-labeled with 32P-orthophosphate or radiolabeled amino acids. The 3 NFP subunits, H(igh), M(iddle), and L(ow), were among a group of axonally transported proteins that incorporated high levels of 32P. Covalent addition of phosphate slowed the electrophoretic mobility of H and M on SDS polyacrylamide gels and shifted the charge of all 3 subunits toward more acidic pH values, thereby providing an index of the phosphorylation state of this radiolabeled population of NFPs. NFPs were extensively phosphorylated before they entered axons at the optic nerve level, and continued to be modified during transport along RGC axons at the optic nerve and tract level. H and M exhibited charge shifts of 0.2-0.6 units toward a more acidic pH during axoplasmic transport. The charge modifications became more prominent when NFPs reached distal axonal levels, which may indicate regional differences in the activity of this modification process along axons. By contrast, the L subunit became more basic in charge, consistent with decreases in the phosphorylation state during transport. Additional observations (Nixon and Lewis, 1986) that a considerable proportion of phosphate groups initially added to L and M were later removed as neurofilaments advanced along RGC axons support the notion that the changing phosphorylation state of NFP subunits during axoplasmic transport reflects a dynamic equilibrium between phosphorylation and dephosphorylation events. Topographical remodeling of phosphate groups on NFPs during axoplasmic transport is proposed as a possible mechanism for coordinating interactions between neurofilaments and other constituents, as these elements are transported and integrated into the axonal cytoskeleton.