The temperature dependence of the single-turnover redox reaction on the electron-donor side of Photosystem II (PS II) has been investigated by measuring light-induced FTIR difference spectra of DCMU-treated PS II membranes at various temperatures of 80–240 K. The negative band at 1404 cm −1 in the difference spectra exhibited the same temperature dependence as the S 2 formation in the oxygen-evolving center previously determined by EPR spectroscopy (De Paula, J.C., Innes, J.B. and Brudvig, G.W. (1985) Biochemistry 24, 8114–8120). This observation strongly supports our previous assignment of the 1404 cm −1 band to the structural change of proteins upon the S 1 → S 2 transition (Noguchi, T., Ono, T. and Inoue, Y. (1992) Biochemistry 31, 5953–5956). The temperature dependence of the 1660 cm −1 band due to cytochrome b 559 (Cyt b 559) oxidation has shown a complementary relationship to that of the 1404 cm −1 band, consistent with the view that the oxidation of Cyt b 559 competes with the S 2 formation on the electron-donor side of PS II. The present FTIR data also suggest that the structural difference between the two S 2 states exhibiting the g = 4.1 EPR signal and the multiline signal is not very large as far as the protein moiety around the Mn cluster responsible for the 1404 cm −1 band is concerned.