Inhibitors of the interaction between the p53 tumor-suppressor protein and its natural human inhibitor HDM2 are attractive as potential anticancer agents. In earlier work we explored designing β-hairpin peptidomimetics of the α-helical epitope on p53 that would bind tightly to the p53-binding site on HDM2. The β-hairpin is used as a scaffold to display energetically hot residues in an optimal array for interaction with HDM2. The initial lead β-hairpin mimetic, with a weak inhibitory activity (IC50=125 μM), was optimized to afford cyclo-(L-Pro-Phe-Glu-6ClTrp-Leu-Asp-Trp-Glu-Phe-D-Pro) (where 6ClTrp=L-6-chlorotryptophan), which has an affinity almost 1000 times higher (IC50=140 nM). In this work, insights into the origins of this affinity maturation based on structure–activity studies and an X-ray crystal structure of the inhibitor/HDM2(residues 17–125) complex at 1.4 Å resolution are described. The crystal structure confirms the β-hairpin conformation of the bound ligand, and also reveals that a significant component of the affinity increase arises through new aromatic/aromatic stacking interactions between side chains around the hairpin and groups on the surface of HDM2.