Initiation of translation on picornavirus RNAs is accomplished through internal binding of ribosomes to a complex cis-acting element. Here we show that efficient function of this element involves two appropriately spaced smaller elements: UUUCC and an AUG. This conclusion emerged from analysis of the genome structures of spontaneous revertants of mutant polioviruses with extended insertions between the UUUCC and AUG motifs. It was confirmed by the results obtained with specially designed constructs. A similarity to the prokaryotic translation initiation mechanism, which involves the Shine-Dalgarno sequence, is emphasized, but in the picornavirus system the position of the UUUCC must be strictly fixed relative to upstream cis-acting elements, and the AUG may not necessarily serve as an initiation codon.