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 structure by fostering cation‐dependent interactions among NF sidearms. By inducing NF bundling, phosphorylation impedes their axonal transport. To examine the impact of the known NF kinase cdk5 on these phenomena, transfected cells with constructs expressing GFP‐tagged NF‐H sidearms (lacking the rod domain to preclude assembly) with and without site‐directed mutagenesis of 7 cdk5 consensus sites, and monitored the impact on NF transport and association with the axonal NF bundle. These mutations did not alter transport but pseudo‐phosphorylated mutants displayed a greater association with axonal NF bundles. By contrast, these same mutations in full‐length NF‐H altered NF transport as well as bundling. Since isolated sidearms cannot assemble, they can only interact with NFs via a single sidearm–sidearm interaction, while assembled NFs can form multiple such interactions. These finding suggest that individual sidearm–sidearm interactions are dynamic and do not persist long enough to slow NF transport, and that bundle formation and maintenance depends upon both the long half‐life of NF polymers and the establishment of multiple phosphorylation‐dependent sidearm‐mediated interactions among NFs. © 2010 Wiley‐Liss, Inc.