Abstract When hepatocytes polarize, a subset of cellular proteins specifically localizes to the apical cell surface forming the boundary of the bile canaliculus. We have isolated a cDNA encoding a protein recognized by a monoclonal antibody (9C5) that specifically stains the bile canaliculus. The encoded protein (Ag-9C5) is a cytoplasmic protein with three leucine zippers and a zinc finger at the C-terminus. Extensive amino acid sequence similarity indicates that Ag-9C5 is likely the chicken homologue of a human protein, FIP2, which interacts with huntingtin and Rab8. Epitope-tagged Ag-9C5 colocalizes with endogenous Ag-9C5 and other canaliculus marker antigens in transfected organ cultures. In Cos7 cells and MDCK cells Ag-9C5 forms punctate cytoplasmic structures. In intact tissues Ag-9C5 is highly concentrated at the apical surfaces of cells that secrete protein from the apical surfaces, but is found in a fine punctate cytoplasmic pattern in other polarized epithelia. Because this protein has a number of characteristics of proteins that act as scaffolds for assembly of protein complexes (e.g., the cytoplasmic domain of classical cadherins and the FERM superfamily of proteins), it appears that FIP2/Ag-9C5 may act as a scaffold for assembling a complex of proteins that are involved in targeting of some secretory vesicles to defined regions of the cell surface.