Neurofilament proteins, the major cytoskeletal components of large myelinated axons, are highly phosphorylated by second messenger-dependent and -independent kinases. These kinases, together with tubulins and other cytoskeletal proteins, have been shown to bind to neurofilament preparations. Cdk5 and Erk2, proline-directed kinases in neuronal tissues, phosphorylate the Lys-Ser-Pro (KSP) repeats in tail domains of NF-H, NF-M, and other axonal proteins such as tau and synapsin. In neurofilament and microtubule preparations from rat brain, we demonstrated by Western blot analysis that cdk5, a neuronal cyclin dependent kinase and Erk1/2 were associated with complexes of NF proteins, tubulins and tau. Using P13(suc1) affinity chromatography, a procedure known to bind cdc2-like kinases in proliferating cells with high affinity, we obtained a P13 complex from a rat brain extract exhibiting the same profiles of cdk5 and Erk2 bound to cytoskeletal proteins. The phosphorylation activities of these preparations and the effect of the cdk5 inhibitor, butyrolactone, were consistent with the presence of active kinases. Finally, during a column fractionation and purification of Erk kinases from rat brain extracts, fractions enriched in Erk kinase activity also exhibited co-elution of phosphorylated NF-H, tubulin, tau and cdk5. We suggest that in mammalian brain, different kinases, their regulators and phosphatases form multimeric complexes with cytoskeletal proteins and regulate multisite phosphorylation from synthesis in the cell body to transport and assembly in the axon.