Regulation of gene expression at the level of mRNA translation is important to rapid alternation of cell metabolism in response to stress. In eukaryotes, regulation of protein synthesis is frequently achieved by the rapid and reversible covalent modification of proteins required for the initiation of translation. This laboratory has studied the role of eIF-2 phosphorylation and its relationship to the accompanying inhibition of protein synthesis. The phosphorylation state of eIF-2 is closely regulated not only by the activity of its specific kinase and protein phospatase, but also by binding of its ligands, GTP and Met-tRNAi and by association with a new polypeptide complex which we designate RF. The major function of RF is to catalyze the exchange of GTP for GDP in the inactive eIF-2.GDP complex, required for reactivation of eIF-2 following each round of initiation. In hemin-deficient lysate, a limited phosphoraylation of eIF-2 (20-30%) is nevertheless able to almost totally inhibit protein synthesis by sequestering the smaller RF pool into an inactive RF.eIF-2(AlphaP) complex, incapable of quanine nucleotide exchange. In addition to alterations of kinase activity, regulation of the phosphorylation state of eIF-2 is controlled by ligand-dependent restrictions on the access of phospatase to phosphorylation sites on eIF-2.