We have previously described the construction of a bicistronic retroviral vector using the picornavirus internal ribosome entry site (IRES), which allows two genes expression simultaneously from a single transcript. This vector transcribes RNA efficiently; however, in some cases the levels of protein production are low. In this report, we further modified the bicistronic vector by abolishing the functional viral gag initiation codon that is retained in the vector at 5' to the first initiation codon of transduced gene. Five different genes, human interleukin 2 (hIL-2), human interleukin 4 (hIL-4), human granulocyte macrophage stimulating factor (hGM-CSF), herpes simplex virus thymidine kinase (HSV-tk) gene, and hepatitis C virus (HCV) core gene (C190), were tested on this modified vector for gene transfer and expression. Our results demonstrated that the new bicistronic vector greatly increased the protein levels when compared with the original one. As the RNA levels and splicing patterns from these two vectors remained similar, the improvement was most likely resulted from the increased translational efficiency.