Herpes simplex virus 2 (HSV-2) strains containing mutations in the virion host shutoff (vhs) protein are attenuated for replication compared with wild-type virus in mouse embryonic fibroblasts (MEFs). However, HSV-2 vhs mutants replicate to near wild-type levels in the absence of the RNA-activated protein kinase (PKR). PKR is one of several kinases that phosphorylates the eukaryotic initiation factor 2α (eIF2α) to inhibit translation initiation, and we previously found that more of the phosphorylated form of eIF2α accumulates in MEFs infected with HSV-2 vhs mutants than with wild-type virus. Here, we show that this increase in phosphorylated eIF2α is primarily PKR dependent. Using MEFs expressing nonphosphorylatable eIF2α, we demonstrate that phosphorylated eIF2α is the primary cause of attenuated replication of HSV-2 vhs mutants and that attenuation correlates with decreased accumulation of viral proteins. Normally, HSV antagonizes eIF2α phosphorylation through the action of ICP34.5, which redirects protein phosphatase 1α (PP1α) to dephosphorylate eIF2α during infection. We show that ICP34.5 does not accumulate efficiently in MEFs infected with HSV-2 vhs mutant viruses, suggesting that the accumulation of phosphorylated eIF2α and the attenuated phenotype of HSV-2 vhs mutants in MEFs result from a deficiency in ICP34.5.