The NS1 protein of influenza A virus binds not only to poly(A) and a stem-bulge region in U6 small nuclear RNA (snRNA), but also to double-stranded (ds) RNA. Binding assays with NS1 protein mutants established that the previously identified RNA-binding domain of the NS1 protein is required for binding to ds RNA as well as for binding to poly(A) and U6 snRNA. In addition, dsRNA competed with U6 snRNA for binding to the NS1 protein, consistent with both RNAs sharing the same binding site on the protein. As a consequence of its binding to dsRNA, the NS1 protein blocks the activation of the dsRNA-activated protein kinase (PKR) in vitro. This kinase phosphorylates the alpha subunit of eukaryotic translation initiation factor 2 (elF-2 alpha), leading to a decrease in the rate of initiation of translation. Assays using purified PKR and purified elF2 demonstrated that the NS1 protein blocks the dsRNA activation of PKR, and experiments using reticulocyte extracts showed that the NS1 protein blocks the inhibition of translation caused by dsRNA activation of PKR. The implications of these results for control mechanisms occurring in influenza virus-infected cells are discussed.