Gas-phase acidities (GA or Delta G(acid)) of acidic di and tripeptides are determined for the first time. The peptides studied are composed of inert alanine (A) residues and one X residue of either aspartic acid (D) or glutamic acid (E): AX, XA, AAX, AXA, and XAA. Experimental GAs were measured by the thermokinetic method of deprotonation ion/molecule reactions in a Fourier transform ion cyclotron resonance mass spectrometer. Calculated GAs were obtained by composite correlated molecular orbital theory at the G3(MP2) level for deprotonation of carboxylic acid groups both at the C-terminus and at the side chain. Excellent agreement was found between experimental and calculated GA values. There is a slight preference for peptides with D being more acidic than analogous peptides with E, which agrees with the GAs of the corresponding amino acids. Experiments showed that peptides are more acidic (lower numerical GA values) when the acidic residue is located at the C-terminus (i.e., AX or AAX). The lowest energy form of deprotonated AAE has a unique structure where the longer side chain of E allows the two carboxylates, which are in close proximity, to share the proton. The tripeptides are less acidic (higher GA value) by 3-7 kcal/mol when the acidic residue is in the center. The tripeptides are more acidic (by 2-10 kcal/mol) than dipeptides containing the same acidic residue at the same location.