The effect of metal cation binding on bacteriorhodopsin (bR) in purple membrane has been examined using in situ attenuated total reflection-Fourier transform infrared difference spectroscopy in aqueous media. It is known that adding metal cations to deionized bR regenerates the purple state from its blue state and recovers the proton pump function. During this process, infrared spectral changes in the frequency region of 1800–1000 cm⁻¹ are monitored. The results reveal that metal cation binding affects the protein conformation, the retinal isomeric composition as well as lipid head groups. It is also observed that metal cation binding induces conformational changes in the α_I-helix region of bR, converting the portion of its α_I-helical domain into β-turn or disordered coil. In addition, the influence of Ho³ binding on the protein and lipid is observed to be larger than that of Ca² . These results suggest that some of the metal cation binding sites are on the membrane lipid domain, while others could be on the intrahelical domain or interhelical loops where the Asp and Glu are located (binding with their COO⁻ groups). Our results also suggest that the removal of the C-terminal of bR increase the accessibility of the binding site of metal cations, which affects protein conformational structure. All these observations are discussed in terms of the two proposals given in the literature regarding the metal cation binding sites.