A panel of mouse B cell hybridomas producing monoclonal antibodies (mAb) directed against rabbit M cell-containing epithelia was developed. By immunohistochemistry, the mAb 5D9, 5B11, 1D9, and 4G2 were found to label approximately 50% of the follicle-associated epithelial (FAE) cell populations overlying lymphoid follicles in Peyer's patches, cecal patch, sacculus rotundus, and appendix. The cell staining was localized to FAE cell basolateral surfaces outlining the M cell pockets which enclosed clusters of mononuclear leukocytes, and extended from the crypts of Peyer's patches and sacculus rotundus, and appendiceal crevices, to the apices of domes. In contrast, the stem cell and proliferative regions facing the lamina propria were devoid of immunologically reactive sites. The mAb 5D9, 1D9, and 4G2 did not recognize antigens associated with non-FAE cells in the intestinal lymphoid tissues examined. Only the mAb 5B11 labeled apical surfaces of Peyer's patch and cecal patch non-FAE. However, this mAb did not label interdomal colonic epithelial cells in sacculus rotundus and appendix. Besides recognizing FAE cells, the mAb 4G2 recognized a cross-reactive antigen displayed by dome and lymphoid follicle lymphocytes. By flow cytometry, the mAb 5D9, 5B11, and 1D9 were shown to stain from 14 to 29% of the cells in M cell-enriched populations prepared from Peyer's patches, sacculus rotundus, and appendix, whereas mAb 4G2 was found to recognize 44-54% of the cells. Two-color flow cytometric analysis showed that the mAb stained a functionally distinct subpopulation of Peyer's patch phagocytic cells and did not recognize spleen macrophages. These findings indicate that the panel of mAb recognized novel antigens expressed by FAE cells overlying intestinal lymphoid aggregates, and that the mAb allow identification of phagocytic M cells in suspensions of FAE cells.