The cyclin-dependent kinase inhibitor p27(Kip) binds to cyclin/cyclin-dependent kinases and preferentially inhibits the catalytic activity of cdk2 and cdk4. The cdk2 binding domain of p27(Kip) was determined using a series of N-terminal truncated and point mutations of GST-p27(Kip). The binding domain was contained within amino acid residues 53-85. The inhibition of the catalytic activity of preformed functional cdk2/cyclin complexes by p27(Kip) was also associated with the binding domain.