A soluble polymerase-template complex prepared from poliovirus-infected cells was found to incorporate radioactive UTP into trichloroacetic acid-insoluble RNA linearly for 8 h in the presence of ATP and Mg2+. Radioactive CTP or GTP was not incorporated under identical conditions. Nearest-neighbor analysis of the in vitro product demonstrated that ATP was added to the viral RNA in the form of polyadenylic acid; UTP was added internally to the 3'-OH group of all four nucelotides. The data can best be explained by the addition of the UTP to the 3'-OH groups of single-stranded breaks in the double-stranded viral RNA and ligation to the adjacent 5'-phosphate groups. The enzymatic activity was also found in encephalomyocarditis virus- and rhinovirus type 1A-infected cells but not in uninfected cells.