We estimated cholesterol in Duchenne muscle plasma membrane using the sterol-specific ligand digitonin. In measuring the extent of digitonin-cholesterol complex formation with freeze-fracture electron microscopy, we found a significantly greater proportion of surface area taken up by complexes in Duchenne muscle (60% +/- 9%) than in controls (36% +/- 4%). This finding suggests that a population of Duchenne muscle fibers has a higher concentration of cholesterol in the plasma membrane. Membrane cholesterol is known to affect membrane deformability and the activity of membrane-bound enzymes and transport proteins; thus a difference in membrane cholesterol might account for structural and functional abnormalities in Duchenne plasma membrane.