Chromoprotein of a light-driven chloride pump, halorhodopsin (HR), was isolated from Halobacterium halobium L-33, which contains HR and "slowly cycling rhodopsin-like pigment" (SR) but lacks bacteriorhodopsin (BR). The isolation was run in the presence of more than 2 M NaCl, which was required to preserve this halophilic retinal protein. Cell envelope vesicles were washed with Tween-20 to remove 80% of the proteins. The residual membranes were solubilized with 0.5% C12E9, which had little effect on the photochemical activities of HR and SR. HR was purified by passing it through a hydroxyapatite and then a phenyl-Sepharose column in 2 M NaCl and 0.5% C12E9. The absorption maximum of HR was 578 nm and the ratio of absorbance at 280 nm to 580 nm was 1.52. The apparent molecular weight of HR was 20,000 on polyacrylamide gel electrophoresis in the presence of SDS. The characteristic, bilobed CD spectrum of HR in the visible region suggested that HR exists as an oligomer in both its membrane-bound and isolated forms.