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Cytoskeletal structures obtained after extraction of Madin-Darby canine kidney epithelial cell monolayers with Triton X-100 were examined in transmission electron micrographs of cell whole mounts and unembedded thick sections. The cytoskeleton, an ordered structure consisting of a peripheral plasma lamina, a complex network of filaments, and chromatin-containing nuclei, was revealed after extraction of intact cells with a nearly physiological buffer containing Triton X-100. The cytoskeleton was further fractionated by extraction with (NH4)2SO4, which left a structure enriched in intermediate filaments and desmosomes around the nuclei. A further digestion with nuclease and elution with (NH4)2SO4 removed the chromatin. The stable structure that remained after this procedure retained much of the epithelial morphology and contained essentially all of the cytokeratin filaments and desmosomes and the chromatin-depleted nuclear matrices. This structural network may serve as a scaffold for epithelial organization. The cytoskeleton and the underlying nuclear matrix intermediate filament scaffold, when examined in both conventional embedded thin sections and in unembedded whole mounts and thick sections, showed the retention of many of the detailed morphological aspects of the intact cells, which suggests a structural continuum linking the nuclear matrix, the intermediate filament network, and the intercellular desmosomal junctions. Most importantly, the protein composition of each of the four fractions obtained by this sequential procedure was essentially unique. Thus, the proteins constituting the soluble fraction, the cytoskeleton, the chromatin fraction, and the underlying nuclear matrix-intermediate filament scaffold are biochemically distinct.