Two-gene vectors with positive or positive-negative drug-selectable markers enable the expansion or elimination of gentetically modified cells in vivo. We have established a bicistronic retroviral vector system which utilizes an internal ribosome entry site (IRES) to co-express two independent genes with high efficiency. As a positive-negative (suicide) marker, Herpes simplex virus thymidine kinase was co-expressed with the human multidrug resistance gene, MDR1. Using this vector, almost all the MDR1-transduced cells showed hypersensitivity to a nucleoside analog, ganciclovir. As a dominant selectable marker, the MDR1 gene was co-expressed with alpha-galactosidase A for the model of gene therapy of Fabry disease. Vincristine selection efficiently enhanced the population of transduced cells expressing the second non-selectable genes. These drug-selectable retroviral vectors could be applicable to the therapy of many diseases.