Leptosphaeria rhodopsin (LR) is an archaeal-type rhodopsin found in fungi, and is the first light-driven proton-pumping retinal protein from eukaryotes. LR pumps protons in a manner similar to that of bacteriorhodopsin (BR), a light-driven proton pump of haloarchaea. The amino acid sequence of LR is more homologous to that of Neurospora rhodopsin (NR) than BR, whereas NR has no proton-pumping activity. These facts raise the question of how the proton-pumping function is achieved. In this paper, we studied structural changes of LR following the retinal photoisomerization by means of low-temperature Fourier transform infrared (FTIR) spectroscopy, and compared the obtained spectra with those for BR and NR. While the light-induced photoisomerization from the all-trans to 13-cis form was commonly observed among LR, BR, and NR, we found that the structural changes of LR are closer to those of BR than to those of NR in terms of detailed vibrational bands of retinal and protein. The most prominent difference was seen for the water O-D stretching vibrations (measured in D2O). LR exhibits an O-D stretch of water at 2257 cm(-1), indicating the presence of a strongly hydrogen-bonded water molecule. Such strongly hydrogen-bonded water molecules (O-D stretch at <2400 cm(-1)) were observed for BR, but not for NR. Comprehensive studies of BR mutants and archaeal rhodopsins have revealed that strongly hydrogen-bonded water molecules are found only in the proteins exhibiting proton-pumping activity, suggesting that strongly hydrogen-bonded water molecules and transient weakening of their binding are essential for the proton-pumping function of rhodopsins. This observation for LR provided additional experimental evidence of the correlation between strongly hydrogen-bonded water molecules and proton-pumping activity of archaeal rhodopsins.