The RNA genome of avian sarcoma virus (ASV) and 38S polyadenylated RNA from cells producing ASV directed the cell-free synthesis of two viral gene products: Pr76gag, the precursor to structural proteins of the virion core, and P180gag/pol, a joint product of two viral genes that is the putative precursor of viral reverse transcriptase. To test the hypothesis that P180gag/pol is synthesized by read-through of the termination codon for gag, we determined the effect of purified yeast suppressor tRNAs on cell-free translation from gag and pol. Although the synthesis of P180gag/pol was not enhanced by the suppressor tRNAs, the use of amber suppressor tRNA reduced the synthesis of Pr76gag and resulted in the synthesis of a new 80, 000 dalton polypeptide (P80) that contained the antigenic determinants of gag. Studies using chemical inhibitors of initiation of translation demonstrated that P80 was generated by the addition of amino acids to the carboxy terminus of Pr76gag. We conclude that the gag gene of ASV is terminated by an amber codon (UAG), and that a second termination signal occurs ∼120 nucleotides downstream. Our data indicate that suppression of a single termination codon does not suffice for the production of P180gag/pol. Thus it is doubtful that suppression by tRNA is responsible for the synthesis of this protein. The mechanism by which P180gag/pol is synthesized in infected cells remains uncertain.