The authors describe a new reverse-genetics system that allows the efficient generation of influenza A viruses entirely from cloned cDNAs. They transfected human embryonic kidney cells (293T) with eight plasmids, each encoding a viral RNA segment of the A/WSN/33 (H1N1) or A/PR/8/34 (H1N1) virus, flanked by the human RNA polymerase I promoter and the mouse RNA polymerase I terminator. This method yielded >1 × 10^5 plaque-forming units (pfu) of virus per ml of supernatant at 48 hours posttransfection. Adding plasmids expressing all remaining viral structural proteins further increased virus production to 3 × 10^4–5 × 10^7 pfu/ml. The system was also used to generate reassortant viruses and viruses with mutations in the PA gene or with a foreign epitope in the neuraminidase protein. This efficient, helper virus-independent system is useful for viral mutagenesis studies and vaccine and gene therapy vector production. The ability to generate infectious RNA viruses from cloned cDNAs has advanced our understanding of pathogens and disease control, but negative-sense RNA viruses have been more challenging to produce. This study overcomes these challenges by generating segmented, negative-sense RNA viruses entirely from cloned cDNAs, which has significant implications for the study of viral life cycles, protein function, and pathogenicity.The authors describe a new reverse-genetics system that allows the efficient generation of influenza A viruses entirely from cloned cDNAs. They transfected human embryonic kidney cells (293T) with eight plasmids, each encoding a viral RNA segment of the A/WSN/33 (H1N1) or A/PR/8/34 (H1N1) virus, flanked by the human RNA polymerase I promoter and the mouse RNA polymerase I terminator. This method yielded >1 × 10^5 plaque-forming units (pfu) of virus per ml of supernatant at 48 hours posttransfection. Adding plasmids expressing all remaining viral structural proteins further increased virus production to 3 × 10^4–5 × 10^7 pfu/ml. The system was also used to generate reassortant viruses and viruses with mutations in the PA gene or with a foreign epitope in the neuraminidase protein. This efficient, helper virus-independent system is useful for viral mutagenesis studies and vaccine and gene therapy vector production. The ability to generate infectious RNA viruses from cloned cDNAs has advanced our understanding of pathogens and disease control, but negative-sense RNA viruses have been more challenging to produce. This study overcomes these challenges by generating segmented, negative-sense RNA viruses entirely from cloned cDNAs, which has significant implications for the study of viral life cycles, protein function, and pathogenicity.