Amide-to-ester backbone mutagenesis enables a specific backbone-backbone hydrogen bond (H-bond) in a protein to be eliminated in order to quantify its energetic contribution to protein folding. To extract a H-bonding free energy from an amide-to-ester perturbation free energy (DeltaG (folding,wt) - DeltaG (folding,mut)), it is necessary to correct for the putative introduction of a lone pair-lone pair electrostatic repulsion, as well as for the transfer free energy differences that may arise between the all amide sequence and the predominantly amide sequence harboring an ester bond. Mutation of the 9-10 amide bond within the V9F variant of the predominantly helical villin headpiece subdomain (HP35) to an ester or an E-olefin backbone bond results in a less stable but defined wild-type fold, an attribute required for this study. Comparing the folding free energies of the ester and E-olefin mutants, with correction for the desolvation free energy differences (ester and E-olefin) and the loss of an n-to-pi* interaction (E-olefin), yields an experimentally based estimate of +0.4 kcal/mol for the O-O repulsion energy in an alpha-helical context, analogous to our previous experimentally based estimate of the O-O repulsion free energy in the context of a beta-sheet. The small O-O repulsion energy indicates that amide-to-ester perturbation free energies can largely be attributed to the deletion of the backbone H-bonds after correction for desolvation differences. Quantitative evaluation of H-bonding in an alpha-helix should now be possible, an important step toward deciphering the balance of forces that enable spontaneous protein folding.