The double-stranded RNA protein kinase (PKR) pathway plays a vital role in the innate immune response to viral infection. Activation of PKR following virus entry can lead to a shutdown in translation, thereby inhibiting viral protein synthesis and replication. Little is currently known about whether human papillomaviruses (HPVs) modulate PKR expression and activity. In this study, normal human foreskin keratinocytes (NHKs) transfected stably with the HPV 31 or 16 genomes and cell lines expressing the HPV 16 E6 and E7 oncoproteins were used to examine effects on the PKR pathway. HPV gene products were found to modulate PKR phosphorylation, activity and localization. The levels of total PKR protein were reduced modestly in cells that maintained HPV 16 or 31 episomes through a reduction in PKR transcription. However, levels of phosphorylated PKR were decreased 4-fold through a post-transcriptional mechanism mediated by E6 and E7 that was independent of the transcriptional downregulation mediated by HPV. In response to infection by vesicular stomatitis virus, phosphorylation of eIF2alpha was blocked in cells expressing HPV oncoproteins, but not in NHKs. Finally, it was observed that the cellular localization of PKR was altered by HPV gene products in HPV raft cultures, as well as HPV-positive patient biopsies. This effect was mediated by the HPV E6 oncoprotein and leads to the co-localization of PKR with P-bodies. These studies demonstrate that high-risk HPVs target the PKR pathway by multiple mechanisms.