The double stranded RNA-dependent protein kinase (PKR), in addition to its role as a translational controlling factor, is a key transcriptional regulator exerting antiviral and antitumoral activities. We have previously shown that induction of NF-kappaB by PKR is involved in apoptosis commitment and this process is mediated through activation of the IKK complex. To gain insights into the mechanism of activation of NF-kappaB by PKR, we have analysed the domains of PKR involved in IKK activation and subsequent NF-kappaB induction. In PKR(0/0) cells infected with a collection of vaccinia virus (VV) recombinants expressing different mutant forms of PKR, we found that only PKR forms conserving the catalytic activity are able to activate NF-kappaB. An inactive PKR mutant (K296R), was unable to induce NF-kappaB activation despite full expression of the protein in a wide range of concentrations, as defined by Western blot, EMSA, IKK kinase activity and NF-kappaB transactivation assays. Moreover, the mutant PKR (K296R) acts as a dominant negative of PKR-induced eIF-2alpha phosphorylation and NF-kappaB activation. However, PKR mutants unable to activate NF-kappaB still retain their ability to associate with the IKK complex, as confirmed by immunoprecipitation analysis. We conclude that the catalytic activity of PKR and not only a protein-protein interaction with the IKK complex, is needed for activation of the transcription factor NF-kappaB.