Cholesterol induces large perturbations in the physical properties of membranes, especially in the structural organization of the phospholipid bilayers and the aggregation and solubility of proteins at physiological temperatures. This study was designed to examine the interaction of cholesterol with lipid and proteins of chloroplasts photosystem II (PSII) submembrane fractions in air dried film at pH 6 -7 with cholesterol concentrations of 0.01 to 20 mM. Fourier transform infrared difference spectroscopy with its self-deconvolution and second derivative methods as well as curve-fitting procedures are used, in order to determine the cholesterol binding mode, the protein conformational changes, and the structural properties of cholesterol-protein complexes. Correlations between the effect of cholesterol on the protein secondary structure and the rate of oxygen evolution in PSII are also established. Spectroscopic evidence showed that at low cholesterol concentration (0.01 and 0.1 mM), minor chol-protein and chol-lipid interactions (through hydrogen bonding) occur with no major perturbations of the protein secondary structure. As cholesterol concentration increases (5 and 10 and 20 mM), major alterations of the protein secondary structure are observed from that of the β£-helix 47% (uncomplexed protein) to 43-39% (complexes) and the β€-sheet structure 18% (uncomplexed protein) to 22-26% (complexes). Those changes coincide with a partial decrease in the rate of the oxygen evolution (8 -33%) is observed in the presence of cholesterol at high concentration.