Replication of the infectious RNA genome of poliovirus is accomplished in cells by the viral RNA polymerase through negative-strand RNA intermediates. Full-length negative-strand poliovirus RNA was synthesized in vitro by transcription of infectious poliovirus cDNA with bacteriophage SP6 DNA-dependent RNA polymerase. When provided with this negative-strand RNA as template, the poliovirus RNA-dependent RNA polymerase synthesized full-length positive-strand molecules. The positive-strand RNAs synthesized in vitro were infectious when transfected into HeLa cells. In contrast, positive-strand copies of poliovirus RNA synthesized in vitro by SP6 polymerase, using a poliovirus cDNA template, were not infectious. Production of infectious positive-strand RNA by the poliovirus polymerase was not observed when magnesium or negative-strand RNA template was omitted from the reaction mixture. Infectivity of the product RNA was not destroyed by DNase treatment. The specific infectivity in HeLa cells of in vitro-synthesized positive-strand RNA was 4 X 10(4) plaque-forming units/micrograms of RNA.