The influenza A virus genome comprises 8 segments of negative-sense RNA that code for up to 12 proteins. RNA segment 2 encodes three proteins, PB1, PB1-F2 and N40, that are translated from the same mRNA by ribosomal leaky scanning and reinitiation. PB1 is a subunit of the trimeric viral RNA polymerase. PB1-F2 has been reported to be a potential virulence factor, and shown to be involved in a number of activities including induction of apoptosis, regulation of viral replication and modulation of the immune response. No function has yet been ascribed to N40 which represents an N-terminally deleted form of PB1. Previous studies on PB1-F2 function mainly used viruses genetically engineered to prevent PB1-F2 expression by mutation of the PB1-F2 start codon. However, ablation of the start codon was shown to increase the expression level of the downstream protein N40. In the present study we generated recombinant A/WSN/33 viruses carrying different combinations of PB1-F2 and N40 knockout mutations. Over-expression of N40 in a PB1-F2 deficient background had a detrimental effect on virus growth in vitro and in vivo. However, ablation of PB1-F2 or N40 expression individually was not disadvantageous for the virus. Primer extension analyses revealed an increase in vRNA production by viruses that over-expressed N40. Our data suggest that the observed attenuation of mutant viruses in vitro and in vivo results from these changes in transcription and replication.