The contribution of backbone hydrogen bonds in alpha-helices to the overall stability of a protein has been examined experimentally by replacing several backbone amide linkages in alpha-helix 39-50 of T4 lysozyme with ester linkages. T4 lysozyme variants wherein the backbone amide bonds between residues Ser38 and Leu39, Lys43 and Leu44, or Ala49 and Ile50 are replaced with ester bonds were generated by incorporating alpha-hydroxy acids at positions 39, 44, or 50, respectively, using unnatural amino acid mutagenesis. The stabilities of the proteins were determined from their thermal denaturation curves as monitored by circular dichroism. Comparison of the thermal stabilities of the amide- and ester-containing proteins shows that the ester substitution has a similar thermodynamic effect at all three positions. At the N- and C-terminal positions, where only one hydrogen-bonding interaction is perturbed, the ester substitution is destabilizing by 0.9 and 0.7 kcal/mol, respectively. Introduction of the ester linkage in the middle of the helix, which alters two hydrogen-bonding interactions, destabilizes the protein by 1.7 kcal/mol. The values obtained from these ester to amide mutations are compared to the values from similar mutations that have been made in other secondary structures and bimolecular complexes.