We have investigated a novel strategy for coexpressing two genes from a retroviral vector. The 5' nontranslated leader region of at least some picornavirus RNAs contains a sequence that can act as an internal ribosome entry site allowing initiation of translation at a downstream AUG codon in a 5' cap-independent manner. To investigate whether such a sequence can function in the context of a retroviral vector, we constructed a spleen necrosis virus-based vector carrying two selectable marker genes separated by the leader region of encephalomyocarditis virus. This vector was genetically stable and efficiently expressed both markers from a single dicistronic transcript. Since the expression of two genes by other strategies in retroviral vectors can often be problematic, these results offer a promising new approach for the design of "double gene" retroviral vectors.