BACKGROUND & AIMS: The follicle-associated epithelium (FAE) of Peyer's patches mainly consists of two cell types: absorptive enterocytes with a brush border and M cells without this apical specialization. To study the controversial ontogeny of M cells (mesenchymal vs. epithelial origin), the expression pattern of tissue-specific cytoskeletal proteins, markers of cell origin that play a crucial role in the specific shape of epithelial cells and brush border assembly, was investigated. METHODS: The localization of cytokeratins, vimentin, and villin was determined on mouse FAE using immunocytochemistry and electron microscopy. RESULTS: Epithelial-specific cytokeratins were expressed in both absorptive enterocytes and M cells, whereas vimentin was not detected in mouse FAE. Villin, a tissue-specific, actin-binding protein of the brush border, was expressed in the two cell types. This protein had an unusual cytoplasmic distribution in FAE cells lacking a brush border and in cells having an intraepithelial pocket filled with lymphocytes. CONCLUSIONS: The presence of villin and the absence of vimentin in M cells support the intestinal origin of M cells. The cytoplasmic distribution of villin provides a new identification criteria for M cells and reflects the reorganization of the F-actin network, which correlates with the inability of M cells to assemble a brush border.