Murine embryonic stem (ES) cells are commonly cultured on feeder layers of primary murine embryonic fibroblasts (MEFs). Because gene targeting experiments often involve sequential selection for multiple-drug resistance in single ES cell lines, we have developed a new mouse strain which represents an economical donor for the production of multiple-drug resistant MEFs. MEFs prepared from the DR-4 mouse strain displayed resistance to concentrations of the drugs G418, 6-thioguanine, puromycin and hygromycin well above those used normally for the selection of drug-resistant ES cells.