Retroviruses package full-length, unspliced RNAs into progeny virions as dimerized RNA genomes. They also use unspliced RNAs as mRNAs to produce the gag and pol gene products. We asked whether a single Rous sarcoma virus (RSV) RNA can be translated and subsequently packaged or whether genomic packaging requires a nontranslated population of RNAs. We addressed this issue by utilizing the translation-dependent nonsense-mediated mRNA decay (NMD) pathway. NMD is the selective destruction of mRNAs bearing premature termination codons (PTCs). The pathway has been shown to be associated with splicing in higher eukaryotes. Here, we demonstrate that both translation and the cellular factor Upf1 are required for the decay of unspliced, PTC-bearing RSV RNA by the NMD pathway. To address the relationship between RNA translation and packaging, we examined virus produced in cells cotransfected with PTC-bearing retroviral clones and wild-type viral clones. We observed that PTC-bearing transcripts are packaged into viral particles at levels three- to fivefold less than those of control RNAs. Since PTC-mediated degradation requires translation, we conclude that RSV can package progeny virion particles using previously translated RNAs.