The junctional complex, including tight junctions (TJs), adherens junctions (AJs), and desmosomes, plays crucial roles in the structure and functions of epithelial cellular sheets. In this study, we evaluated the fluorescence localization-based retrovirus-mediated expression cloning (FL-REX) method as an approach to identify novel molecular components of TJs and AJs. Using an expression library of cDNA-GFP-fusions derived from mRNA of a mouse epithelial cell line, we confirmed that cDNAs for various known TJ- and AJ-components could be cloned in the FL-REX. Furthermore, cDNAs for ARHGAP12 and SPAL3, two putative GTPase activating proteins (GAPs) for small G proteins, were cloned as novel components of the junctional complex. Immunofluorescence staining using antibodies generated in-house demonstrated that these GAPs were localized at epithelial cell-cell junctions in various mouse tissues, and were specific to AJs when observed under confocal laser-scanning microscopy. These data suggest that FL-REX is a powerful tool to identify novel proteins localized at TJs and AJs.