The small GTPase Rac and its effectors, the Pak1 and p35/Cdk5 kinases, have been assigned important roles in regulating cytoskeletal dynamics in neurones. Our previous work revealed that the neuronal p35/Cdk5 kinase associates with Pak1 in a RacGTP dependent manner, causing hyperphosphorylation and downregulation of Pak1 kinase activity. We have now demonstrated direct phosphorylation of Pak1 on Threonine 212 by the p35/Cdk5 kinase. In neuronal growth cones Pak1 phosphorylated on T212 localised to actin and tubulin rich areas suggesting a role in regulating growth cone dynamics. The expression of a non-phosphorylatable Pak1 mutant (Pak1A212) induced dramatic neurite disorganisation. We also observed a strong association between p35/Cdk5 and the Pak1 C-terminal kinase domain. Overall, our data show that in neurones membrane associated, active Pak1 is regulated by the p35/Cdk5 kinase both by association and phosphorylation which is essential for the proper regulation of the cytoskeleton during neurite outgrowth and remodelling.