We have isolated and characterized the light-driven proton pump Bop I from the ultrathin square archaeon Haloquadratum walsbyi, the most abundant component of the dense microbial community inhabiting hypersaline environments. The disruption of cells by hypo-osmotic shock yielded Bop I retinal protein highly enriched membranes, which contain one main 27 kDa protein band together with a high content of the carotenoid bacterioruberin. Light-induced pH changes were observed in suspensions of Bop I retinal protein-enriched membranes under sustained illumination. Solubilization of H. walsbyi cells with Triton X-100, followed by phenyl-Sepharose chromatography, resulted in isolation of two purified Bop I retinal protein bands; mass spectrometry analysis revealed that the Bop I was present as only protein in both the bands. The study of light/dark adaptations, M-decay kinetics, responses to titration with alkali in the dark and endogenous lipid compositions of the two Bop I retinal protein bands showed functional differences that could be attributed to different protein aggregation states. Proton-pumping activity of Bop I during the photocycle was observed in liposomes constituted of archaeal lipids. Similarities and differences of Bop I with other archaeal proton-pumping retinal proteins will be discussed.