Calcium depletion from the endoplasmic reticulum inhibits protein synthesis and correlates with increased phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF-2 alpha) by a mechanism that does not require ongoing protein synthesis. To elucidate whether protein synthesis inhibition requires eIF-2 alpha phosphorylation and whether eIF-2 alpha phosphorylation is mediated by the double-stranded RNA-dependent protein kinase (PKR), we studied protein synthesis in response to calcium depletion mediated by calcium ionophore A23187 in cell lines overexpressing wild-type eIF-2 alpha, a mutant eIF-2 alpha (S51A) that is resistant to phosphorylation, or a dominant negative mutant PKR (K296P in catalytic subdomain II). Expression of either mutant eIF-2 alpha or mutant PKR partially protected NIH3T3 cells from inhibition of protein synthesis upon A23187 treatment. In contrast, overexpression of wild-type PKR increased sensitivity to protein synthesis inhibition mediated by A23187 treatment. In a COS-1 monkey cell transient transfection system, increased eIF-2 alpha phosphorylation in response to A23187 treatment was inhibited by expression of the dominant negative PKR mutant. Overexpression of the PKR regulatory RNA binding domain, independent of the PKR catalytic domain, was sufficient to inhibit increased phosphorylation of eIF-2 alpha upon A23187 treatment. In addition, overexpression of the HIV TAR RNA binding protein also inhibited eIF-2 alpha phosphorylation upon A23187 treatment. Taken together, our data show that calcium depletion activates PKR to phosphorylate eIF-2 alpha, and this activation is likely mediated through the PKR RNA binding domain.