The gas-phase acidities (GAs) of six tripeptides (GlyGlyGly, GlyAlaGly, AlaGlyAla, AlaAlaAla, AibAibAib, and SarSarSar) and their methyl esters were obtained by proton transfer reactions in a Fourier transform ion cyclotron resonance mass spectrometer and G3(MP2) molecular orbital theory calculations. All six peptides have GAs in the range 321.0-323.7 kcal/mol. Their deprotonation to produce [M - H](-) occurs at the C-terminal carboxylic acid group. The tripeptides are about 10 kcal/mol more acidic than the amino acids glycine (Gly) and alanine (Ala). This is consistent with the extensive hydrogen bonding that was found in the tripeptide structures. For the methyl esters, deprotonation occurs at the peptide backbone. G3(MP2) calculations show that the most energetically favored site of deprotonation is an amide nitrogen, with the central amide being generally preferred. Nitrogen deprotonation requires 10-20 kcal/mol less energy than deprotonation at a methylene carbon. Only three of the methyl esters (GlyGlyGly-OMe, GlyAlaGly-OMe, and AlaAlaAla-OMe) deprotonate experimentally by electrospray ionization. Experimental GAs for these esters are in the range of 336.7-338.1 kcal/mol, in excellent agreement with the calculated G3(MP2) values. Experimental GAs could not be obtained for the other three methyl esters (AlaGlyAla-OMe, AibAibAib-OMe, and SarSarSar-OMe) because they did not produce sufficient deprotonated molecular ions. Trisarcosine methyl ester, SarSarSar-OMe, cannot be deprotonated at a central amide nitrogen because methyl groups are present at these sites; consequently, it has a high G3(MP2) GA value (less acidic) of 350.6 kcal/mol for deprotonation at the N-terminal nitrogen. For AlaGlyAla-OMe and AibAibAib-OMe, calculations of van der Waals and solvent accessible surfaces reveal that methyl groups are blocking the amide nitrogen sites. Therefore, conformational and steric hindrance effects are limiting the ability of these peptide methyl esters to deprotonate in the mass spectrometer.