AbstractNeurofilaments (NFs) associate with each other and with other cytoskeletal elements to form a lattice that supports the mature axon. Phosphorylation contributes to formation of this stationary population of NFs by fostering cation‐dependent interactions among NF sidearms. Association of NFs with the stationary phase indirectly competes with NF axonal transport by withdrawing NFs from kinesin‐dependent motility along microtubules. We therefore hypothesized that inhibition of anterograde NF transport may increase incorporation into the stationary phase. To test this hypothesis, we treated differentiated NB2a/d1 cells expressing GFP‐tagged NF subunits with monastrol, a specific inhibitor of kinesin‐5. Monastrol significantly inhibited anterograde axonal transport of NF‐H but not NF‐M, and increased the incorporation of newly‐transported NF subunits into axonal NF bundles. These findings support the notion that NF transport and bundling exert opposing forces on axonal NF dynamics, and that inhibition of anterograde transport of NFs can increase their incorporation into the stationary phase. © 2012 Wiley Periodicals, Inc