Rhodopsin was transferred, as a protein-lipid complex, in its unbleached state from retinal rod outer segments (ROS) into organic solvents by a novel technique not involving detergents. The optical absorbance properties of rhodopsin were examined in (a) the organic solvent and in (b) the protein-lipid complex hydrated after solvent evaporation, (a) In hexane and in ether the difference spectra between dark and irradiated samples show λ_max at 499 ± 1 nm and isosbestic points around 415 ± 4 nm. Bleached rhodopsin in hexane, but not in ether, when supplemented with 9-ris-retinal, regenerated isorhodopsin with a yield of better than 90%. (b) Rhodopsin from ether extracts was deposited in a filter paper disk (Whatman), the solvent evaporated, and then the complex hydrated. At room temperature rhodopsin showed absorption spectra in the dark and after bleaching similar to those recorded in ROS membranes; on addition of ll-c/s-retinal to the filter paper, regeneration of rhodopsin to ~95% was obtained. When rhodopsin was bleached at -196 °C and spectra were recorded as a function of temperature (from -196 to 20 °C), the expected displacements of Xmax due to the production of the intermediates (batho, lumi, meta I, meta II, and meta III) were observed. The photochemical functionality of this preparation was also evaluated by measuring the kinetics of the metarhodopsin I to metarhodopsin II transition simultaneously at 478 and 380 nm, respectively. The temperature and pH dependencies of this reaction were comparable to the values reported for rhodopsin in ROS membranes. The kinetics of this photochemical conversion were also recorded in hexane and ether. The results indicate that rhodopsin derived from hexane and ether extracts conserves the basic spectral features it displays in native retinal ROS disk membranes and, therefore, constitutes an active starting point for reconstitution studies in planar bilayers and other model membranes.