Initiation of translation in picornavirus RNAs occurs internally, mediated by an internal ribosome entry site (IRES) element. This property has been exploited to coexpress proteins from a single bicistronic transcription unit in eukaryotic cells. The region that separates the IRES element from the authentic initiator codon of the second gene plays an important role in the translation efficiency of this cistron. In the present report, we have analyzed the effect of sequence modifications in this region on the translation efficiency directed by the foot-and-mouth disease (FMDV) IRES in bicistronic expression vectors. Insertion of various sequences, which contained additional start codons and/or the capacity to form hairpins immediately downstream of the 3' border of the IRES, strongly reduced the translation efficiency of the second gene in bicistronic RNAs. Interestingly, an increase of distance per se did not have a deleterious effect on translation efficiency. The bicistronic vector studied here tolerated 95 nucleotides between the 3' border of the IRES and the authentic start codon, provided that out-of-frame AUG codons or hairpins were not present in this RNA segment. These results indicate that FMDV-derived bicistronic constructs are extremely well suited for use in eukaryotic expression vectors.