We show that phosphorylation of the alpha subunit of eukaryotic translation initiation factor 2 (eIF-2) by the protein kinase GCN2 mediates translational control of the yeast transcriptional activator GCN4. In vitro, GCN2 specifically phosphorylates the alpha subunit of rabbit or yeast eIF-2. In vivo, phosphorylation of eIF-2 alpha increases in response to amino acid starvation, which is dependent on GCN2. Substitution of Ser-51 with alanine eliminates phosphorylation of eIF-2 alpha by GCN2 in vivo and in vitro and abolishes increased expression of GCN4 and amino acid biosynthetic genes under its control in amino acid-starved cells. The Asp-51 substitution mimics the phosphorylated state and derepresses GCN4 in the absence of GCN2. Thus, an established mechanism for regulating total protein synthesis in mammalian cells mediates gene-specific translational control in yeast.