A cytopathic mutant of Rous sarcoma virus-PrB was isolated and shown to have two large deletions, which result in the junction of Gag sequences in P27 to the 5′ end of Env, and in the loss of the Src gene. This replication-defective (rd) and transformation-defective (td) mutant can replicate in the presence of its helper, which is also td, but the viral particles produced are poorly infectious. Most of the virions do not contain viral RNA, and the mutant RNA accumulates in infected cells, where it is poorly translated and packaged. Molecular clones of the mutant, of its helper and of a PrBtd strain were obtained in λ-EMBL3, characterized, shown to be biologically active by transfection assays and sequenced. Nucleotide sequence comparisons indicate that the strong ribosome-binding site of Rous sarcoma virus RNA, responsible for the efficient RNA translation in vivo and in vitro, is mutated in PrB-(HM) mutant RNA; this causes the inhibition of RNA translation, as demonstrated by translation competition experiments using virus RNA made in vitro that carries the original or the mutated ribosome-binding site. In addition, an insertion present at the 3′ end of both the mutant and the helper RNA, but absent in PrBtd RNA, is probably responsible for the inhibition of RNA packaging. Finally, these data are discussed in the light of a model of a 5′ → 3′ Rous sarcoma virus RNA structure leading to a circular RNA molecule, which has implications in RNA translation, packaging and reverse transcription.