The conformational stability of the beta-hairpin miniprotein, CLN025, a variant of chignolin in which the N- and C-terminal glycines are replaced by tyrosines, in various concentrations of guanidinium chloride (GdmCl) and urea was examined by molecular dynamics simulations and electronic circular dichroism (ECD) spectropolarimetry. The peptide maintains its beta-hairpin conformation in GdmCl and urea solutions. In GdmCl, Gly7 influences the turn to reduce the number of Asp3-Gly7 H-bonds and the Tyr1-Trp9 H-bond is lost. The structure of the peptide is less stable in 3M GdmCl than in water or 6 M GdmCl, because the number of Asp3-Thr8 and Tyr1-Tyr10 H-bonds are reduced and the Tyr2 side chain moves away from the Pro4 and Trp9 side chains and toward the Tyr10 side chain. This reduces the number of Tyr2-Pro4 CH-pi interactions and Tyr2-Trp9 and Tyr1-Tyr10 aromatic-aromatic (Ar-Ar) interactions and increases the number of Tyr2-Tyr10 Ar-Ar interactions. In 6 M GdmCl at 300 and 333 K, the number of Tyr1-Tyr10 and Asp3-Thr8 H-bonds increases, but fewer structures have Tyr2-Pro4 CH-pi and Tyr1-Tyr10 and Tyr2-Trp9 Ar-Ar interactions. In urea, Gly7 is in a mixture of beta-turn and random meander structures and the number of Asp3-Thr6 and Tyr1-Tyr10 H-bonds are reduced as are the number of Tyr2-Pro4 CH-pi interactions and Tyr1-Tyr10 and Tyr2-Trp9 Ar-Ar interactions. In 4 M urea, a shorter turn places Gly7 into the beta-sheet region and Tyr10 is pushed out into the solvent. In 8 M urea, the number of Asp3-Glu5 H-bonds is increased and the beta-sheet is lost, but the electrostatic interaction between the charged termini is restored and a cation-pi interaction between the indolyl ring of Trp9 and the positively charged N-terminus is formed. In 8 M urea at 333 K, the beta-hairpin conformation is almost lost. The structure of CLN025 is stable, because the weakly polar interactions and H-bonds maintain the beta-hairpin conformation in the various environments. CLN025 should not be considered a miniprotein, because it lacks a well-defined tertiary structure, it is resistant to denaturation, it does not have an increased heat capacity near its melting temperature, and the structures near and above the melting temperature retain a beta-hairpin conformation.