Stable transfection of mammalian cells is a widely used technique for the study of gene expression and protein purification. However, selection of cell lines expressing desired genes from a large number of candidate clones is often labor-intensive and time consuming. To improve the efficiency of stable cell line production, we have used a bicistronic mammalian expression vector, pIRES1hyg, which contains the internal ribosome entry site (IRES) of the encephalomyocarditis virus (ECMV). The IRES element permits the translation of two open reading frames from one messenger RNA: one reading frame encoding the recombinant protein of interest and the other an antibiotic resistant marker (e.g. hygromycin). We demonstrate that the use of the bicistronic vector significantly facilitates the creation of stable mammalian cell lines, because all selected antibiotic-resistant colonies express the recombinant gene of interest. Therefore, the use of the pIRES1hyg bicistronic vector for stable transfection eliminates the need to screen large numbers of colonies to find functional clones. We conclude that the IRES bicistronic vector provides a powerful tool for efficient selection of stable transformants in mammalian cells.