PKR is an interferon(IFN)-induced, serine-threonine protein kinase, which plays a crucial role in IFN's antiviral and antiproliferative actions. The three known activators of PKR are dsRNA, heparin, and PACT. PACT activates PKR by direct protein-protein interaction in response to cellular stress. The human TAR (trans-activating region)-binding protein (TRBP), which is very homologous to PACT, also interacts with PKR, leading to an inhibition of PKR activity. Since these two highly homologous proteins have opposite effects on PKR, we examined if they interact with PKR differently by assaying their interaction with various point mutants of PKR. Our results indicate that TRBP and PACT interact with PKR through the same residues, and no differences were identified in these two interactions. Domain swap experiments between PACT and TRBP indicated that the inhibitory effects of TRBP on PKR activity are mediated through its carboxy-terminal residues, which contain TRBP's third dsRNA-binding motif.