The cytoskeleton of colon (11 cases) and breast adenocarcinomas (9 cases) was characterized with the use of immunohistochemistry on tissue sections and one- and two-dimensional (2-D) gel electrophoresis of cytoskeletal extracts of tumor cells. By immunofluorescence, antibodies to epidermal cytokeratin (CK) and Mallory body CK recognized cytoplasmic filaments +/- desmosomal contacts, respectively, in both colon and breast adenocarcinomas. In addition, cytoskeletal extracts of both tumors showed similar CK polypeptides by 2-D gel electrophoresis. By immunoperoxidase, anti-actin antibody stained the apical margin of tumor cells in eight (73%) colon adenocarcinomas and four of five metastases, while diffuse cytoplasmic staining was seen in only one (9%) breast adenocarcinoma and not in five metastases. With 2-D gel electrophoresis, a cytoskeletal-associated doublet polypeptide was found in seven (64%) colon adenocarcinomas but not in the breast adenocarcinomas. By immunoblotting, the doublet did not consist of CK polypeptides, vimentin, or type IV collagen. These findings may facilitate the differentiation of colon and breast adenocarcinomas.