Villin is a 95-kilodalton gastrointestinal-related cytoskeletal protein associated with axial microfilament bundles of brush border microvilli. We tested the hypothesis that expression of this protein was restricted to adenocarcinomas of gastrointestinal origin in a retrospective study of 203 human neoplasm that had been fixed in Carnoy's or methacarn fixative and embedded in paraffin. Villin expression was restricted to a subset of epithelial tumors, with no expression noted in any cases of sarcoma, melanoma, or lymphoma. Villin proved to be a very sensitive and relatively specific marker of gastrointestinal adenocarcinomas: all colonic, gastric, and pancreatic carcinomas were positive, and none of the breast or lung carcinomas were positive, with the single exception of a bronchioalveolar adenocarcinoma. However, a subset of nongastrointestinal adenocarcinomas, including some adenocarcinomas of ovary, endometrium, and kidney, were also positive. Nonetheless, at least three distinct patterns of villin expression were discerned, some of which were quite specific for individual tumor types. Thus, an apical or brush border pattern of villin expression was seen in virtually all gastrointestinal adenocarcinomas, as well as in the occasional villin-positive endometrial or ovarian adenocarcinomas. A membranous distribution of carcinomas, mimicked the patterns of the normal counterpart tissue, e.g., delineation of bile canalicular structures. Finally, no relationship was found between the presence, or pattern of expression, of villin and the state of tumor differentiation. It is concluded that antibodies to villin may play an important role in immunocytochemical analyses of poorly differentiated malignant tumors in appropriately fixed, paraffin-embedded tissue and in cases where the primary site is indeterminant from clinical history and histology.