Protein kinase PKR is activated in mammalian cells during viral infection, leading to phosphorylation of the alpha subunit of eukaryotic initiation factor-2 (eIF-2alpha) and inhibition of protein synthesis. This antiviral response is thought to be mediated by association of double-stranded RNA (ds-RNA), a by-product of viral replication, with two ds-RNA-binding domains (DRBDs) located in the amino terminus of PKR. Recent studies have observed that expression of mammalian PKR in yeast leads to a slow growth phenotype due to hyperphosphorylation of eIF-2alpha. In this report, we observed that while DRBD sequences are required for PKR to function in the yeast model system, these sequences are not required for in vitro phosphorylation of eIF-2alpha. To explain this apparent contradiction, we proposed that these sequences are required to target the kinase to the translation machinery. Using sucrose gradient sedimentation, we found that wild-type PKR was associated with ribosomes, specifically with 40 S particles. Deletions or residue substitutions in the DRBD sequences blocked kinase interaction with ribosomes. These results indicate that in addition to mediating ds-RNA control of PKR, the DRBD sequences facilitate PKR association with ribosomes. Targeting to ribosomes may enhance in vivo phosphorylation of eIF-2alpha, by providing PKR access to its substrate.