Design, synthesis and characterization of CHAPSTEROL, a novel cholesterol-based detergent developed for functional solubilization of cholesterol-dependent membrane proteins are described. To validate CHAPSTEROL, we employed the oxytocin receptor, a G protein-coupled receptor requiring cholesterol for its high-affinity binding state. Using the photoactivatable cholesterol analogue [3H]6,6-azocholestan-3beta-ol[3alphaH], we demonstrate that solubilization by CHAPSTEROL leads to an enrichment of cholesterol-binding proteins whereas the widely used bile acid derivative CHAPSO leads to a significant depletion of cholesterol-binding proteins. Similar to Triton X-100 and CHAPS, CHAPSTEROL maintains the localization of caveolin as well as cholesterol and sphingomyelin to lipid rafts, i.e. detergent-insoluble microdomains of the plasma membrane. The data suggest that CHAPSTEROL is an appropriate detergent for the solubilization of cholesterol-dependent membrane proteins and isolation of rafts.