RNA signals at the ends of the genes of respiratory syncytial (RS) virus direct polyadenylation and termination of viral transcription. These gene ends contain two conserved regions, a pentanucleotide and a tract of uridylate (U) residues, separated by an A/U-rich central region that is less well conserved. The U tract is thought to be the template for polyadenylation of viral mRNAs by reiterative transcription. The cis-acting requirements for termination were investigated by mutagenesis of the matrix (M) gene end (3'-UCAAUUAUUUUUU-5') in a dicistronic RNA replicon. Termination efficiencies were quantitated by intracellular metabolic labeling of monocistronic mRNAs and the dicistronic readthrough RNAs that result when termination fails to occur. All three regions of the gene end were necessary for termination. Mutation of each of the first 8 nucleotides of the M gene end to all other nucleotides showed that nucleotides 2 to 6 were important for termination and intolerant of change, whereas nucleotides 1 and 7 were tolerant of change. At position 8, A or U allowed termination, but G or C did not. Both the length and the position of the U tract were important for termination. U residues at positions 9 to 12 were necessary, while additional U residues at position 8, and especially position 13, enhanced termination efficiency. Altering the length of the central region abolished termination, suggesting that the position of the U tract with respect to the 3'-UCAAU-5' sequence was critical. The termination efficiencies of each of the 10 genes of RS virus are different. Since transcription is obligatorily sequential and termination of each gene is required for transcription of the next gene downstream, these differences may contribute to gene regulation. In agreement with our data, the naturally occurring gene ends of RS virus that terminate inefficiently have short U tracts or other sequence features that correlated with decreased termination when similar mutations were analyzed in RNA replicons.