Cyclin-dependent kinases (CDKs) that control cell cycle progression are regulated in many ways, including activating phosphorylation of a conserved threonine residue. This essential phosphorylation is carried out by the CDK-activating kinase (CAK). Here we examine the effects of replacing this threonine residue in human CDK2 by serine. We found that cyclin A bound equally well to wild-type CDK2 (CDK2 Thr-160 ) or to the mutant CDK2 (CDK2 Ser-160 ). In the absence of activating phosphorylation, CDK2Ser-160 -cyclin A complexes were more active than wild-type CDK2 Thr-160 -cyclin A complexes. In contrast, following activating phosphorylation, CDK2Ser-160 -cyclin A complexes were less active than phosphorylated CDK2Thr-160 -cyclin A complexes, reflecting a much smaller effect of activating phosphorylation on CDK2Ser-160 . The kinetic parameters for phosphorylating histone H1 were similar for mutant and wild-type CDK2, ruling out a general defect in catalytic activity. Interestingly, the CDK2 Ser-160 mutant was selectively defective in phosphorylating a peptide derived from the C-terminal domain of RNA polymerase II. CDK2 was efficiently phosphorylated by CAKs, both human p40 MO15 (CDK7)-cyclin H and budding yeast Cak1p. In fact, the k cat values for phosphorylation of CDK2 were significantly higher than for phosphorylation of CDK2 Thr-160 , indicating that CDK2 Ser-160 is actually phosphorylated more efficiently than wild-type CDK2. In contrast, dephosphorylation proceeded more slowly with CDK2Ser-160 than with wild-type CDK2, either in HeLa cell extract or by purified PP2C. Combined with the more efficient phosphorylation of CDK2 Ser-160 by CAK, we suggest that one reason for the conservation of threonine as the site of activating phosphorylation may be to favor unphosphorylated CDKs following the degradation of cyclins.