The subgenomic mRNA's of Rous sarcoma virus share a common 5' leader sequence spliced from genomic RNA. We have examined the 5' terminal sequences of four Rous sarcoma virus RNAs: virion RNA and three species of intracellular mRNA which direct the synthesis of the RSV gene products. The lengths of the leaders on the RNAs were determined by the extent that they could protect cloned Rous sarcoma virus DNA fragments from S1 nuclease digestion after RNA-DNA hybridization. We found that the subgenomic mRNA's that direct the synthesis of the env and src gene products have uninterrupted spliced leader sequences of approximately 390 nucleotides, whereas virion RNA and full-length intracellular viral RNA have 5' termini homologous to the cloned viral DNA probe over at least the first 735 bases. In the accompanying manuscript we have determined the nucleotide sequence of the 5' end of the Rous sarcoma virus genome, including the candidate splice donor site identified here (Swanstrom et al., J. Virol 41:535-541, 1982).