Fodrin (nonerythroid spectrin) is a 475,000 molecular weight (MW) (apparent) heterodimeric actin-binding protein usually found in mature cells at the cytoplasmic face of the plasma membrane. While its precise role is uncertain, it may participate in the establishment and/or maintenance of cell polarity, shape, and specialized receptor domains. In polarized epithelial cells, an asymmetric distribution of fodrin appears to signal phenotypic maturity. Using immunohistochemical techniques, the distribution of fodrin in enterocytes during normal crypt-to-villus maturation, and in adenomas, adenocarcinomas, and cultured Madin-Darby Canine Kidney (MDCK) cells has been studied and its abundance quantitated by immunoblotting and digital immunofluorescent confocal microscopy. During normal maturation, fodrin was found to assemble at the apex of the enterocyte, presumably in the terminal web, only in those cells near the villus tip. Villin was found in an apical location in both crypt and surface enterocytes. In adenocarcinomas of the colon (n = 11), there were enhanced levels of fodrin at the apex, and an approximately threefold increase in the total amount of fodrin per cell relative to normal crypt enterocytes. An increased percentage of this protein was also found in the cytoplasm. Adenomas (n = 7), nonconfluent MDCK cells in culture, and two (of two) cases of ductal carcinoma of the breast also demonstrated enhanced cytoplasmic and total fodrin. Supranormal levels of fodrin at the apex of enterocytes were also observed in Crohn's disease samples and in the normal-appearing enterocytes adjacent to a tumor. It is hypothesized that increased apical fodrin may signal a reaction of the microvillar brush border to pathologic stress, while increased cytoplasmic and total pools of fodrin may mark neoplastic activity. These findings may be of diagnostic value, particularly in the evaluation of small biopsies or cytologic material.