Protein targeting from cytosol into the endoplasmic reticulum (ER) in mammalian cells is an initial step in the biogenesis of most secretory and membrane proteins as well as the proteins localized in the ER, Golgi, and lysosomes. Identification of these proteins is crucial for understanding this biological process, which varies in different mammalian cell types. To identify ER-targeted proteins, we have developed a method for high-throughput screening of genes that encode proteins transported into the ER in living mammalian cells. The principle is based on the reconstitution of two fragments of split green fluorescent protein (GFP) by protein splicing and retrovirus-mediated expression cloning. The method is able to collect ER-targeted proteins systematically and to accurately identify many novel proteins. This method will facilitate understanding of the secretory pathway and intercellular communication in living mammalian cells.