An amide-to-ester backbone substitution in a protein is accomplished by replacing an alpha-amino acid residue with the corresponding alpha-hydroxy acid, preserving stereochemistry, and conformation of the backbone and the structure of the side chain. This substitution replaces the amide NH (a hydrogen bond donor) with an ester O (which is not a hydrogen bond donor) and the amide carbonyl (a strong hydrogen bond acceptor) with an ester carbonyl (a weaker hydrogen bond acceptor), thus perturbing folding energetics. Amide-to-ester perturbations were used to evaluate the thermodynamic contribution of each hydrogen bond in the PIN WW domain, a three-stranded beta-sheet protein. Our results reveal that removing a hydrogen bond donor destabilizes the native state more than weakening a hydrogen bond acceptor and that the degree of destabilization is strongly dependent on the location of the amide bond replaced. Hydrogen bonds near turns or at the ends of beta-strands are less influential than hydrogen bonds that are protected within a hydrophobic core. Beta-sheet destabilization caused by an amide-to-ester substitution cannot be directly related to hydrogen bond strength because of differences in the solvation and electrostatic interactions of amides and esters. We propose corrections for these differences to obtain approximate hydrogen bond strengths from destabilization energies. These corrections, however, do not alter the trends noted above, indicating that the destabilization energy of an amide-to-ester mutation is a good first-order approximation of the free energy of formation of a backbone amide hydrogen bond.