The NS1 proteins of influenza A and B viruses (A/NS1 and B/NS1 proteins) have only approximately 20% amino acid sequence identity. Nevertheless, these proteins show several functional similarities, such as their ability to bind to the same RNA targets and to inhibit the activation of protein kinase R in vitro. A critical function of the A/NS1 protein is the inhibition of synthesis of alpha/beta interferon (IFN-alpha/beta) during viral infection. Recently, it was also found that the B/NS1 protein inhibits IFN-alpha/beta synthesis in virus-infected cells. We have now found that the expression of the B/NS1 protein complements the growth of an influenza A virus with A/NS1 deleted. Expression of the full-length B/NS1 protein (281 amino acids), as well as either its N-terminal RNA-binding domain (amino acids 1 to 93) or C-terminal domain (amino acids 94 to 281), in the absence of any other influenza B virus proteins resulted in the inhibition of IRF-3 nuclear translocation and IFN-beta promoter activation. A mutational analysis of the truncated B/NS1(1-93) protein showed that RNA-binding activity correlated with IFN-beta promoter inhibition. In addition, a recombinant influenza B virus with NS1 deleted induces higher levels of IRF-3 activation, as determined by its nuclear translocation, and of IFN-alpha/beta synthesis than wild-type influenza B virus. Our results support the hypothesis that the NS1 protein of influenza B virus plays an important role in antagonizing the IRF-3- and IFN-induced antiviral host responses to virus infection.