The S1 species of mammalian reovirus mRNA, like a number of other viral but not cellular mRNAs, codes for two dissimilar polypeptides by initiation of translation at two 5'-proximal, out-of-frame AUG codons. To determine if uninfected cells can utilize bicistronic genes, a bovine papilloma virus-based vector system was used to select mouse C127 cell lines containing multiple integrated copies of the reovirus S1 gene. These cell lines produced both reovirus polypeptides from a single mRNA. In addition, studies of COS cells transfected with the S1 gene containing small changes around the first AUG suggest that bicistronic mRNA translation is regulated at the level of elongation. A model is proposed in which ribosomes engaged in translation of one reading frame interfere with movement of ribosomes in the other frame because of differences in codon usage. Expression of bicistronic genes may be similarly regulated in virus-infected cells.