To investigate cytoskeletal changes associated with axonal regrowth from damaged nerve cells in the mammalian CNS, we examined the slow transport of axonal proteins during the regeneration of adult rat retinal ganglion cell (RGC) axons. Although normally such RGC axons do not regrow after injury in the CNS, they can extend several centimeters when their nonneuronal environment is changed by replacing the optic nerve (ON) with a grafted segment of peripheral nerve (PN). Proteins transported in axons of RGCs from intact control and PN-grafted animals were labeled by an intraocular injection of 35S-methionine and examined 4–60 days later by SDS PAGE. During RGC regeneration into PN grafts, the transport rate of tubulin and neurofilament increased twofold, whereas that of actin decreased to nearly one third of its normal rate. Thus, in these regenerating RGC axons, all three major cytoskeletal proteins were largely transported within a single rate component rather than in the two separate components (SCa and SCb) normally observed in the intact ON. Furthermore, the 200 kDa neurofilament protein (NF-H) was persistently detected in Western blots during periods of active regeneration, a finding that contrasts with the late appearance of the NF-H during the developmental growth of retinal axons. The changes in slow transport observed during RGC regeneration in adult rats may reflect growth-associated responses of mature CNS neurons during periods of active axonal extension.