Previously we found that the amino-terminal region of the NS1 protein of influenza A virus plays a key role in preventing the induction of beta interferon (IFN-beta) in virus-infected cells. This region is characterized by its ability to bind to different RNA species, including double-stranded RNA (dsRNA), a known potent inducer of IFNs. In order to investigate whether the NS1 RNA-binding activity is required for its IFN antagonist properties, we have generated a recombinant influenza A virus which expresses a mutant NS1 protein defective in dsRNA binding. For this purpose, we substituted alanines for two basic amino acids within NS1 (R38 and K41) that were previously found to be required for RNA binding. Cells infected with the resulting recombinant virus showed increased IFN-beta production, demonstrating that these two amino acids play a critical role in the inhibition of IFN production by the NS1 protein during viral infection. In addition, this virus grew to lower titers than wild-type virus in MDCK cells, and it was attenuated in mice. Interestingly, passaging in MDCK cells resulted in the selection of a mutant virus containing a third mutation at amino acid residue 42 of the NS1 protein (S42G). This mutation did not result in a gain in dsRNA-binding activity by the NS1 protein, as measured by an in vitro assay. Nevertheless, the NS1 R38AK41AS42G mutant virus was able to replicate in MDCK cells to titers close to those of wild-type virus. This mutant virus had intermediate virulence in mice, between those of the wild-type and parental NS1 R38AK41A viruses. These results suggest not only that the IFN antagonist properties of the NS1 protein depend on its ability to bind dsRNA but also that they can be modulated by amino acid residues not involved in RNA binding.