The glycosylphosphatidylinositol (GPI)-anchored proteins are widely distributed in eukaryotic cells, from yeasts to mammals. A number of proteins, such as glycosyltransferases, are necessary for GPI anchor biosynthesis. Cloning of genes encoding these proteins is required for analyses of their nature and the biosynthetic pathway. Here we report a new method of expression cloning that is applicable to many mutant rodent and human cells, and its application for cloning a human cDNA termed PIG-F (for Phosphatidyl-Inositol-Glycan class F) using a Thy-1-negative mutant murine thymoma cell line of complementation class F. PIG-F takes a part in the step of transfer of ethanolamine phosphate to the GPI intermediate containing three residues of mannose. This expression cloning strategy is applicable to the identification of not only other genes involved in GPI anchor biosynthesis but also human disease-associated genes using mutant mammalian cell lines.