A method is reported for the isolation of desmosomes in a high yield and of a purity suitable for biochemical analysis. The procedure utilizes the selective solubilizing action of citric acid-sodium citrate (CASC) buffer, pH 2.6, on the non-cornified layers of cow nose epidermis, followed by discontinuous sucrose density gradient centrifugation. Electron microscopy with both thin sections of pellets and unfixed spread preparations reveals that after centrifugation, desmosomes are located mainly at the 55–60% sucrose interface. In the desmosome preparation thus obtained, the characteristic desmosome structure is well preserved, showing the midline, unit membranes, and dense plaques. Furthermore, removal of the epidermal filament bundles by the solubilizing action of CASC buffer has revealed a finely filamentous layer on the cytoplasmic surface of the plaques. The dimensions, location, and appearance of this layer correspond with those of the "connecting component" which has been previously suggested as being responsible for the attachment of epidermal filament bundles to the desmosome.