Cation–π interactions are common in proteins, but their contribution to the stability and specificity of protein structure has not been well established. In this study, we examined the impact of cation–π interactions in a diagonal position of a β-hairpin peptide through comparison of the interaction of Phe or Trp with Lys or Arg. The diagonal interactions ranged from −0.20 to −0.48 kcal/mole. Our experimental values for the diagonal cation–π interactions are similar to those found in α-helical studies. Upfield shifting of the Lys and Arg side chains indicates that the geometries of cation–π interactions adopted in the 12-residue β-hairpin are comparable to those found in protein structures. The Lys was found to interact through the polarized Cε, and the Arg is stacked against the aromatic ring of Phe or Trp. Folding of these peptides was found to be enthalpically favorable (ΔH° ∼ −3 kcal/mole) and entropically unfavorable (ΔS°∼ −8 cal mole−1 K−1).