Bovine rhodopsin was purified from n-octylglucoside-solubilized retinas by high-performance size-exclusion chromatography. In one chromatographic step, six protein fractions were separated with baseline resolution. The major fraction was identified as monomeric rhodopsin by absorption spectroscopy. Amino acid analysis of this fraction further supported the assignment. A comparison of the elution profiles of rhodopsin purified by this method with that purified by Concanavalin A-Sepharose 4B affinity chromatography suggested that rhodopsin from high-performance chromatography was slightly purer than the conventionally purified rhodopsin.