Sequences of avian retroviral RNAs suggest that short open reading frames in the putatively untranslated leader sequences might direct the synthesis of small peptides. Previous analyses indicate that translation of Rous sarcoma virus (RSV) RNA in vitro faithfully reflects translation of the viral RNA in the chick cell. Accordingly, we sought to determine if the heptapeptide LP1, encoded in the open reading frame closest to the 5′ end of RSV RNA, could be synthesized in vitro since this would strongly suggest that it might also be synthesized in vivo. Here we confirm that RSV RNA directs the synthesis of LP1 in rabbit reticulocyte lysates. LP1 is rapidly degraded in the lysate by an aminopeptidase activity. On the basis of the following observations, we propose that the open reading frame encoding LP1 plays a role in the life cycle of avian retroviruses. (1) The LP1 open reading frame is ubiquitious with respect to position and length in 12 strains of avian retrovirus. (2) In the amino acid sequences of the 12 strains, only three of the seven residues are invariant. (3) On the basis of the conservation of the -3 and +4 nucleotides flanking the AUG codon, the strengths of initiation for translation of LP1 are approximately the same in the different viruses. (4) The LP1 open reading frame is positioned in front of sites on retrovirus RNA that are required for initiation of cDNA synthesis and for packaging of the RNA into mature virus.