The incorporation of the eight gene segments of influenza A virus in virions, either through a random or a specific mechanism, has been under debate for many years. Using reverse genetics techniques and trans-complementation of viral proteins, we showed that the production of viruses containing seven gene segments is very inefficient, which can be overcome by providing the 8th gene segment in a non-functional form. We conclude that sequences in the 5' and 3' ends of the polymerase gene segments contain packaging signals. Our methods will facilitate the further mapping of the packaging signals of influenza A virus.