Fluorescence quenching of membrane-bound fluorophors by nitroxide spin-labeled phospholipids has proved to be a useful technique for examining lipid-fluorophor contact (1-3). When the fluorophor is the tryptophanyl residue of a polypeptide, this method can yield relative binding constants of phospholipids to a membrane-bound polypeptide. When a spin-labeled phospholipid loses contact with a tryptophanyl residue, tryptophanyl fluorescence increases. Converse behavior occurs in a bilayer lipid mixture containing spin-labeled lipid, unlabeled lipid, and a membrane protein. There, as the unlabeled lipid separates from the rest of the mixture, fluorescence decreases while the environment of the fluorophors becomes enriched in the quenching, spin-labeled phospholipid. For example, in a bilayer vesicle containing a spin-labeled phosphatidylcholine, dipalmitoylphosphatidylcholine (DPPC), and the membrane-bound polypeptide gramicidin A', as the temperature is lowered below the DPPC gel-liquid crystal