A fraction enriched in cell-to-cell adherens and tight junctions was isolated from the chick liver, and the undercoat-constitutive proteins were extracted from this isolated junctional fraction. Monoclonal antibodies (mAbs) were then obtained by injecting this extract into rats, and five antigens were identified to be concentrated in the isolated junctional fraction. We have characterized one mAb (E14 mAb) and its antigen (E14). By immunoblotting of the isolated junctional fraction the E14 mAb reacted strongly with a single band of approximately 155 kDa, and E14 was highly concentrated in the isolated junctional fraction. Immunofluorescence microscopy revealed that the E14 mAb exclusively stained the junctional complex region of the liver, renal epithelial cells, and the cell-cell border of endothelial cells in various tissues. The intercalated disc of the heart was also significantly stained. However, the E14 signal was hardly detected from intestinal epithelial cells. By immunoelectron microscopy using renal epithelial cells, E14 was mainly detected in the fibrous structures associated with the cell-to-cell adherens junction. We conclude that E14 is a novel undercoat-constitutive protein found in certain types of cell-to-cell adherens junctions.