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Molecular patterns in pathogenic RNAs can be recognized by the innate immune system, and a component of this response is the interferon-induced enzyme RNA-activated protein kinase (PKR). The major activators of PKR have been proposed to be long double-stranded RNAs. We report that RNAs with very limited secondary structures activate PKR in a 5′-triphosphate–dependent fashion in vitro and in vivo. Activation of PKR by 5′-triphosphate RNA is independent of RIG-I and is enhanced by treatment with type 1 interferon (IFN-α). Surveillance of molecular features at the 5′ end of transcripts by PKR presents a means of allowing pathogenic RNA to be distinguished from self-RNA. The evidence presented here suggests that this form of RNA-based discrimination may be a critical step in mounting an early immune response.