1. Retinal isomers extracted from the acid-hydrolysate of cetyltrimethylammonium bromide-treated dark-adapted bacteriorhodopsin (bRD) were analyzed in a high performance liquid chromatograph (HPLC) system. The extract from bRD contains almost equal molar amounts of both 13-cis retinal and all-trans retinal isomers. The extent of isomerization and the yield of both isomers during the isolation process were investigated by the application of the same extraction procedure to artificial bacteriorhodopsin reconstituted with 13-cis retinal isomer (13-cis bacteriorhodopsin) and also to light-adapted bacteriorhodopsin (bRL) which has been shown to contain only the all-trans isomer (all-trans bacteriorhodopsin). 2. A reconstituted bacteriorhodopsin, which had been prepared from apo-bacteriorhodopsin and an equimolar mixture of both 13-cis retinal and all-trans retinal isomers, showed an absorption spectrum having the same maximum wavelength as that of bRD even at the beginning of the reconstitution process. 3. Analysis of the photosteady states of bRD at -190 degrees C revealed that it was composed of two different species, one having 13-cis retinal and the other having all-trans retinal isomers in approximately equal molar amounts. These two also gave their respective photoproducts. 4. From these results it can be concluded that bRD contains both 13-cis retinal and all-trans retinal isomers in nearly equal molar amounts as its chromophore.