O⁶-Substituted guanines are adenosine 5‘-triphosphate (ATP) competitive inhibitors of CDK1/cyclin B1 and CDK2/cyclin A, the O⁶ substituent occupying the kinase ribose binding site. Fifty-eight O⁶-substituted guanines were prepared to probe the ribose pocket, and the structures of four representative compounds bound to monomeric CDK2 were determined by X-ray crystallography. Optimum binding occurs with a moderately sized aliphatic O⁶ substituent that packs tightly against the hydrophobic patch presented by the glycine loop, centered on Val18, an interaction promoted by the conformational restraints imposed in a cyclohexylmethyl or cyclohexenylmethyl ring. Structure-based design generated (R)-(2-amino-9H-purin-6-yloxymethyl)pyrrolidin-2-one (56), which reproduces the reported hydrogen bonds formed between ATP and Asp86 and Gln131 but failed to improve inhibitory potency. Thus, the parent compound O⁶-cyclohexylmethylguanine (NU2058, 25) is the preferred starting point for exploring other areas of the kinase active site.