The hepatitis C virus (HCV) envelope glycoprotein-2 inhibits the interferon (IFN)-induced, double-stranded RNA-activated protein kinase (PKR) via the PKR eukaryotic initiation factor-2alpha phosphorylation homology domain (PePHD). The present study examined the genetic variability of the PePHD in patients receiving IFN therapy. The PePHD from 12 HCV genotype 1 (HCV-1)-infected patients receiving daily IFN therapy was amplified by reverse-transcriptase polymerase chain reaction and analyzed by direct and clonal sequencing. The PePHD was highly conserved in 38 HCV GenBank isolates. There was no difference in pretreatment PePHD sequences isolated from IFN responders versus nonresponders. The major PePHD quasi-species variant did not change after 6 weeks of daily IFN therapy, and in 1 patient the major quasi-species variant did not change during 9 months of observation. Sequencing of 25 pretreatment PePHD clones from 3 patients confirmed that there was extremely low sequence variability surrounding the PePHD. The PePHD is highly conserved in HCV-1-infected IFN responders and nonresponders and does not appear to evolve in response to IFN therapy.