We have developed a new vector for the stable expression of recombinant protein in mammalian cells. In this vector, designated pEFIRES-P, both the recombinant cDNA and the puromycin resistant gene (pac) are transcribed as a single message driven by the strong human polypeptide chain elongation factor 1alpha promoter. The presence of an internal ribosome entry site ensures any clones that are resistant to puromycin also express high levels of recombinant protein encoded by cDNA cloned upstream from the pac gene. We report the use of this vector to generate stable cell lines expressing human pyruvate carboxylase and show that following transfection and clonal selection, all twenty colonies that conferred high dose resistance to puromycin also expressed very high levels of functional pyruvate carboxylase. Use of pEFIRES-P should permit the rapid and efficient generation of stable cell lines for the characterisation of recombinant proteins.