Protein kinase R (PKR) is a central component of the interferon antiviral defense pathway. Upon binding double-stranded RNA (dsRNA), PKR undergoes autophosphorylation reactions that activate the kinase. PKR then phosphorylates eukaryotic initiation factor 2alpha, thus inhibiting protein synthesis in virally infected cells. Using a series of dsRNAs of increasing length, we define the mechanism of PKR activation. A minimal dsRNA of 30 bp is required to bind two PKR monomers and 30 bp is the smallest dsRNA that elicits autophosphorylation activity. Thus, the ability of dsRNAs to function as PKR activators is correlated with binding of two or more PKR monomers. Sedimentation velocity data fit a model where PKR monomers sequentially attach to a single dsRNA. These results support an activation mechanism where the role of the dsRNA is to bring two or more PKR monomers in close proximity to enhance dimerization via the kinase domain. This model explains the inhibition observed at high dsRNA concentrations and the strong dependence of maximum activation on dsRNA binding affinity. Binding affinities increase dramatically upon reducing the salt concentration from 200 to 75 mM NaCl and we observe that a second PKR can bind to the 20-bp dsRNA. Nonspecific assembly of PKR on dsRNA occurs stochastically without apparent cooperativity.