The initiation of encephalomyocarditis virus RNA translation is by internal ribosome entry almost exclusively at the 11th AUG codon from the 5'-end, which is the central of the three AUG codons in the sequence. ..ACGAUGAUAAUAUGGCCACAACCAUG.., and is located some 25 nt downstream from an oligopyrimidine tract conserved amongst related viruses. As the sequences between the oligopyrimidine tract and AUG-10/11 are poorly conserved and thus possibly serve only as a spacer, the influence of this spacer length on initiation frequency at the three AUG codons was examined in vitro and in vivo. Deletion of 11 residues resulted in initiation almost exclusively at AUG-12 but at significantly reduced overall efficiency. Insertion of eight residues caused a 15-fold increase in initiation frequency at AUG-10 and a decrease at AUG-11. Longer insertions reduced overall efficiency without changing the initiation site preferences. With the wild-type spacing, complete substitution of the oligopyrimidine tract by purines caused a 30-35% decrease in initiation efficiency, and partial substitution only a 10-15% decrease. Thus the internal initiation mechanism selects the initiation site partly on the basis of its distance from upstream elements, of which the oligopyrimidine tract is not the most critical, but for reasons not yet understood a preference for AUG-11 is superimposed on this selection.