The activation of cyclin-dependent kinases (CDKs) requires the phosphorylation of a conserved threonine (Thr(160) in Cdk2) by CDK-activating kinase (CAK). Human KAP (also called Cdl1), a CDK-associated phosphatase, was shown to dephosphorylate Thr(160) in human Cdk2. KAP was unable to dephosphorylate Tyr(15) and only dephosphorylated Thr(160) in native monomeric Cdk2. The binding of cyclin A to Cdk2 inhibited the dephosphorylation of Thr(160) by KAP but did not preclude the binding of KAP to the cyclin A-Cdk2 complex. Moreover, the dephosphorylation of Thr(160) bit KAP prevented Cdk2 kinase activity upon subsequent association with cyclin A. These results suggest that KAP binds to Cdk2 and dephosphorylates Thr(160) when the associated cyclin subunit is degraded or dissociates.