Cyclin-dependent kinase 5 (Cdk5) is activated by the neuronal-specific activator protein, p35. In contrast to the activation of typical CDKs by cyclin subunits, p35.Cdk5 was not further activated by the CDK-activating kinase (CAK) and was neither phosphorylated nor inhibited by the Tyr-15-specific Wee1 kinase. The previously identified proteolytic active fragment of p35, p25 (residues 91-307) as well as the slightly smaller fragment containing residues 109-291, was found to be sufficient to bind and activate Cdk5. Other CDKs, including Cdk2, associated weakly with p25. However, their kinase activity was only activated to the low level observed for cyclin A.Cdk2 without Thr-160 phosphorylation, and phosphorylation of Thr-160 in Cdk2 did not activate the p25.Cdk2 complex further. We have identified distinct regions in p35 required for binding to Cdk5 or activation of Cdk5. Residues approximately 150-200 of p35 were sufficient for binding to Cdk5, but residues approximately 279-291 were needed in addition for activation of Cdk5 in vitro.