Intramolecular hydrogen bonding and conformation of tripeptide amines in CDCl3 solutions have been examined by variable-temperature FTIR spectroscopy. Absorption in the NH stretching region of N-acetyl-L-Pro-L-Leu-Gly-NH2 was decomposed into some component bands by least-squares fitting. Two broad bands at around 3350 and 3290 cm(-1) were assigned to intramolecularly hydrogen-bond-free NHs of the three different amide groups by referring to spectra of analogous amide compounds. From temperature dependence of the band intensities, it is concluded that the C-terminal amide group takes part in the hydrogen bonding in both hydrogen-bonded conformers. From comparison with spectra of N-trifluoroacetyl-L-Pro-L-Leu-Gly-NH2, the 3290 cm(-1) band has been assigned to a 13-membered ring with the hydrogen bond between NH of the C-terminal amide group and CO of the N-terminal acetyl group. The 3350-cm(-1) band, on the other hand, has been assigned to a 10-membered hydrogen-bonded ring between the C-terminal amide NH and a prolyl carbonyl group. A van’t Hoff analysis shows that the 13-membered hydrogen-bonded ring is enthalpically more favorable but entropically less favorable than the 10-membered hydrogen-bonded ring.