The activation of most protein kinases requires phosphorylation at a conserved site within a structurally defined segment termed the activation loop. A classic example is the regulation of the cell cycle control enzyme, CDK2/cyclin A, in which catalytic activation depends on phosphorylation at Thr 160 in CDK2. The structural consequences of phosphorylation have been revealed by x-ray crystallographic studies on CDK2/cyclin A and include changes in conformation, mainly of the activation loop. Here, we describe the kinetic basis for activation by phosphorylation in CDK2/cyclin A. Phosphorylation results in a 100,000-fold increase in catalytic efficiency and an approximate 1,000-fold increase in the overall turnover rate. The effects of phosphorylation on the individual steps in the catalytic reaction pathway were determined using solvent viscosometric techniques. It was found that the increase in catalytic power arises mainly from a 3,000-fold increase in the rate of the phosphoryl group transfer step with a more moderate increase in substrate binding affinity. In contrast, the rate of phosphoryl group transfer in the ATPase pathway was unaffected by phosphorylation, demonstrating that phosphorylation at Thr 160 does not serve to stabilize ATP in the ATPase reaction. Thus, we hypothesize that the role of phosphorylation in the kinase reaction may be to specifically stabilize the peptide phosphoacceptor group. Cellular proliferation is controlled by a family of protein kinases in which the catalytic subunits are members of the cyclin-dependent kinase (CDK) 1 family and the regulatory subunits are cyclins. To date, nine distinct CDKs in addition to eight different cyclins have been identified, in which different CDK/cyclin combinations serve to regulate distinct points in the mammalian cell division cycle. Although Cdc2 (CDK1)/ cyclin B controls the transition of cells from the G 2 to M-phase, the activities of CDK2/cyclin E and CDK2/cyclin A are critical for G 1 /S-phase transition and progression through S-phase, respectively (1). Since the critical role of the CDKs in cell cycle control has been well established, understanding the details of their regulation is now of fundamental importance.The three-dimensional structures of several forms of CDK2 have been solved by x-ray crystallography. Like all protein kinases, CDK2 displays a globular fold consisting of two lobes, a smaller N-terminal lobe that is principally ␤-sheet and a larger C-terminal lobe that is principally ␣-helix. The bilobal interface constitutes the active site cleft into which the adenine ring of substrate ATP is deeply buried. The ATP ␥-phosphate is directed toward the mouth of the active site where peptide and protein substrates bind and where phosphoryl group transfer occurs (for a review see Ref.2). Located near the mouth of the active site is a conserved loop structure termed the activation loop (residues 146 -166). This loop structure is present in all protein kinases (3), and phosphorylation at a conserved site within the activation loop ...