Isomerization of all-trans to 11-cis retinol has been studied in a membrane preparation from the nuclear fraction of bovine retinal pigment epithelium. When the nuclear membrane preparation deprived of endogenous retinoids is incubated with 4.5 microM all-trans-retinol, the mean value calculated for the isomerase activity is 1.32 nmol 11-cis retinol formed hr-1 mg protein-1. Simultaneous formation of all-trans and 11-cis retinyl esters is also observed in the nuclear preparation. When assayed under the same experimental condition, RPE 150,000 g post-nuclear sediment shows about 70% of the isomerase activity found in the nuclear membrane fraction. Treatment of the nuclear membrane fraction with 0.5% (w/v) CHAPS produces a 200,000-g supernatant retaining 80% of the total isomerase activity and leads to a modest purification of the enzyme activity. Apparent values for Km and Vmax of the solubilized enzyme are 1.6 microM and 2.5 nmol 11-cis retinol formed h-1 mg protein-1, respectively. Bovine serum albumin and beta-lactoglobulin effectively stimulate the isomerization reaction. The mechanism underlying this activating effect remains unclarified at present. Some hypotheses are discussed.