To determine the stability of individual hydrogen bonds in the helical ion channel peptide alamethicin in methanol, pH-dependent hydrogen-deuterium exchange rates were measured using nuclear magnetic resonance (NMR) spectroscopy. pH-dependent exchange data were also measured for the protected dipeptides N-acetyl-K-X-NH2, where X is G, A, or Aib (alpha-aminoisobutyric acid) and N-acetyl-A-A-NH2 to characterize exchange parameters for non-hydrogen-bonded amides in methanol. The pH-dependent amide exchange data for alamethicin and the dipeptides were fit to an equation defining acid and base catalysis by solvent. Electrostatic effects arising from the peptide helix macrodipole are shown to affect (by up to 20-fold) the acid- and base-catalyzed exchange rate constants for amides at the helix termini. Amide exchange protection factors for the alamethicin amides were determined either from the ratio of k(min)/k(min(free)) (where k(min), and k(min(free)) are the exchange rate constants at the minimum of the pH-dependent plots for alamethicin amides, and a non-hydrogen-bonded amide, respectively) or by correcting acid- and base-catalyzed protection factors for sequence-dependent inductive and steric effects. The enhancement of acid-catalyzed, relative to base-catalyzed, exchange protection factors supports the imidic acid mechanism for acid-catalyzed exchange (Perrin, C. L. Acc. Chem Res. 1989, 22, 268-275) for alamethicin in methanol. The exchange protection factors are a measure of the stability of each of the hydrogen bonds of alamethicin with respect to exchange-limiting backbone fluctuations, and indicate that the peptide has stable hydrogen bonds throughout the helical structure. The high exchange stability of Aib3 and V15 NH’s indicates that these amides are protected by 3(10) helical hydrogen bonds. In contrast to melittin (Dempsey, C. E. Biochemistry 1992, 31, 4705-4712) the P14 residue of alamethicin does not markedly disrupt hydrogen bonding around the center of the alamethicin helix. The exchange data demonstrate that an internal proline residue may have quite different effects on the structure and dynamics of an a-helix depending on the peptide sequence around it.