Many viruses use stop codon readthrough as a strategy to produce extended coat or replicase proteins. The stop codon of the barley yellow dwarf virus (PAV serotype) coat protein gene is read through at a low rate. This produces an extended polypeptide which becomes part of the virion. We have analyzed the cis-acting sequences in the barley yellow dwarf virus PAV genome required for this programmed readthrough in vitro in wheat germ extracts and reticulocyte lysates and in vivo in oat protoplasts. Two regions 3' to the stop codon were required. Deletion of sections containing the first 5 of the 16 CCN NNN repeats located 3' of the stop codon greatly reduced readthrough in vitro and in vivo. Surprisingly, readthrough also required a second, more distal element that is located 697 to 758 bases 3' of the stop codon within the readthrough open reading frame. This element also functioned in vivo in oat protoplasts when placed more than 2 kb from the coat protein gene stop in the untranslated region following a GUS reporter gene. This is the first report of a long-range readthrough signal in viruses.