As phosphorylation of eukaryotic translation initiation factor 2α (eIF2α) on Ser51 inhibits protein synthesis, cells restrict this phosphorylation to the antiviral protein kinase PKR and related eIF2α kinases. In the crystal structure of the PKR-eIF2α complex, the C-terminal lobe of the kinase contacts eIF2α on a face remote from Ser51, leaving Ser51 ∼ 20 Å from the kinase active site. PKR mutations that cripple the eIF2α-binding site impair phosphorylation; here, we identify mutations in eIF2α that restore Ser51 phosphorylation by PKR with a crippled substrate-binding site. These eIF2α mutations either disrupt a hydrophobic network that restricts the position of Ser51 or alter a linkage between the PKR-docking region and the Ser51 loop. We propose that the protected state of Ser51 in free eIF2α prevents promiscuous phosphorylation and the attendant translational regulation by heterologous kinases, whereas docking of eIF2α on PKR induces a conformational change that regulates the degree of Ser51 exposure and thus restricts phosphorylation to the proper kinases.