Xanthorhodopsiri (xR) is a member of the retinal protein family and acts as a proton pump in the cell membranes of the extremely halophilic eubacterium Salinibacter ruber. In addition to the retinal chromophore, xR contains a carotenoid, which acts as a light harvesting antenna as it transfers 40% of the quanta it absorbs to the retinal. Our previous studies have shown that the CD and absorption spectra of xR arc dramatically affected clue to, the protonation of two different residues. It is still unclear whether xR can bind Cations. Electron paramagnetic resonance (EPR) spectroscopy used in the present study revealed that xR can bind divalent cations, such as Mn2+ and Ca2+, to deionized xR (DI-xR). We also demonstrate that xR can bind 1 equiv of Mn2+ to a, high-affinity binding site followed by binding of similar to 40 equiv in cooperative manner and similar to 100 equiv of Mn2+ that are weakly bound. SQUID magnetic studies suggest that the high cooperative binding of Mn2+ cations to xR is due to the formation of Mn2+ clusters. Our data demonstrate that Ca2+ cations bind to DI-xR with a lower affinity than Mn2+,supporting the assumption that binding of Mn2+ occurs through cluster formation, because CA(2+) cations cannot form clusters in Contrast to Mn2+.