The protonation sites and conformations of protonated glycine and its peptides (Gly(1-5)) have been investigated using infrared multiple photon dossociation (IRMPD) spectroscopy in combination with theoretical calculations. For small peptides, protonation is generally presumed to occur at the amine nitrogen of the N-terminus or a nitrogen of a basic side chain. However, for triglycine, the experimental and calculated results indicate that one of the main species is an isomer in which the proton is bound to an amide oxygen. The amide II vibrational mode is found to be very sensitive to the protonation site. When the protonation site is at the amine nitrogen, the amide II mode appears around 1540 cm(-1) for diglycine, tetraglycine, pentaglycine; and one of the main isomers of triglycine (GGGH02). When the proton is bound to an amide oxygen, the amide II mode is blue-shifted to 1590 cm(-1), as seen in GGGH01. IR spectra have been obtained to provide direct evidence that an amide oxygen may serve as the protonation site in a peptide. An analogous result is found for the tripeptide of alanine. In the progression from glycine to pentaglycine, the corresponding conformations of the most stable isomers vary from linear to cyclic structures. Both glycine and diglycine are linear structures, while the most stable isomers of the tetra- and pentapeptides are both cyclic structures. For triglycine, the linear and cyclic isomers are found to coexist. The carbonyl stretches also directly reflect the conformational changes. For the linear isomers of the di- and tripeptides of glycine, two well-separated bands are observed. The amide I modes appear slightly above 1700 cm(-1), but as a result of the fact that the C=O bond in the carboxylic acid moiety is stronger than those of the amide carbonyls, the corresponding band appears near 1800 cm(-1). However, for the cyclic isomers of the tri-, tetra-, and pentapeptides, the carbonyl oxygen in the carboxylic acid group acts as a proton acceptor to form a very strong intramolecular hydrogen bond with the protonated amine terminus. This results in a weakening of the C=O bond, such that the amide I modes are nearly identical in frequency to the carbonyl stretch of the carboxylic acid group.