The solvent effect on the conformational equilibria of [Leu] 5 -enkephalin was investigated using FT-IR spectroscopy. FT-IR spectra of the amide I region of [Leu] 5 -enkephalin were decomposed into several component bands using a curve fitting method. Infrared bands of the β-turn, 3 10 -helix, and the extended conformers were observed in deuterium oxide ( 2 H 2 O) solution, while the band of the 3 10 -helix was not observed in dimethylsulfoxide (DMSO). From the temperature dependence of the relative integrated intensity ratio, we determined the enthalpy differences between the conformers (the β-turn, 3 10 -helix, and extended conformers). The enthalpy level is the β-turn<the 3 10 -helix<the extended conformer in the order of the stability, and that in DMSO is the β-turn<the extended conformer. We also determined the enthalpy differences between the conformers in the gas phase by ab initio molecular orbital method. We decomposed the enthalpy difference into two contributions from the intramolecular and the solute–solvent interactions in order to discuss the solvent effects on the conformational equilibria. Based on these results, we found that the strength order of the hydrogen bonds is: the intramolecular hydrogen bond<the peptide⋯ 2 H 2 O<the peptide⋯DMSO. We discuss the enthalpy levels of the conformations in both solutions in terms of the energy balance between the intramolecular and intermolecular hydrogen bonds.