Neurofilaments have been proposed to regulate axonal stability and diameter through changes in number and subunit composition. We have found that pathway and target innervation directly influence the molecular composition of neurofilaments within regenerating optic axons of Xenopus laevis. Immunocytochemistry was used to examine neurofilaments within two abnormal visual pathways. The first was an aberrant, transient retinoretinal projection, which formed when some axons entered the contralateral optic nerve at the chiasm. The second was formed by regenerating axons deprived of their normal targets by surgical ablation of both optic tecta. Distal to an orbital nerve crush, the neurofilament proteins NF-L, NF-M, NF-H, and XNIF disappear from degenerating fibers. In normally regenerating axons, these neurofilament proteins emerge in a progression reminiscent of development. In the aberrant retinoretinal projection, levels of XNIF, NF-L, and -M remained lower than in normally regenerating axons, whereas NF-H and a phosphorylated form of NF-M were undetectable for at least 35 d after nerve crush. Normally, these two latter forms reappear between 15 and 21 d after surgery. Thus, this transient, incorrect axonal projection expressed neurofilaments in a very different pattern from correctly regenerating axons. In tecta-ablated frogs, staining of phosphorylation independent epitopes of XNIF, NF-L, and -M increased normally after axons entered the tract, but that of NF-H and phosphorylated NF-M remained low for at least 42 d after axotomy. Thus, separate parts of the visual pathway influence the complexity of neurofilaments.