Neurofilaments are composed of three polypeptide subunits (NF-H, NF-M and NF-L). They are the most abundant cytoskeletal element in large myelinated axons and play a central role in development of axonal caliber. To perform this role, neurofilaments are transported from their site of synthesis, the cell bodies, to the distal axons. Previous studies showed that overexpression of NF-M in transgenic mice led to accumulation of neurofilaments in neurons and a reduction in the number of neurofilaments in axons, suggesting that axonal transport of neurofilaments was slowed. To determine whether this was the case, we measured axonal transport velocities in the wild type and transgenic mice overexpressing NF-M by the classical pulse-labeling method using 35S-methionine. We found that neurofilament transport in peripheral motor axons can be described with a model consistent with two linear velocities. Contrary to expectations, both velocities were accelerated by overexpression of NF-M. These results suggest that subunit composition in neurofilaments play a regulatory role in neurofilament transport. In addition, these results show that there are regional differences in neurofilament transport along long axons and these differences may be the basis for selective regional accumulation of neurofilaments in various neurological disorders.