Purine nucleotides are structural components of the genetic material, function as phosphate donors, participate in cellular signaling, are cofactors in enzymatic reactions, and constitute the main carriers of cellular energy. Thus, imbalances in A/G nucleotide biosynthesis affect nearly the whole cellular metabolism and must be tightly regulated. We have identified a substitution mutation (G388D) that reduces the activity of the GMP synthase Gua1 in budding yeast and the total G-nucleotide pool, leading to precipitous reductions in the GDP/GTP ratio and ATP level in vivo. gua1-G388D strongly reduces the rate of growth, impairs general protein synthesis, and derepresses translation of GCN4 mRNA, encoding a transcriptional activator of diverse amino acid biosynthetic enzymes. Although processing of pre-tRNA(i)(Met) and other tRNA precursors, and the aminoacylation of tRNA(i)(Met) are also strongly impaired in gua1-G388D cells, tRNA(i)(Met)-containing complexes with the macromolecular composition of the eIF2·tRNA(i)(Met.)GTP complex (TC) and the multifactor complex (MFC) required for translation initiation accumulate ∼10-fold in gua1-G388D cells and, to a lesser extent, in wild-type (WT) cells treated with 6-azauracil (6AU). Consistently, addition of an external supply of guanine reverts all the phenotypes of gua1-G388D cells, but not those of gua1-G388D Δhpt1 mutants unable to refill the internal GMP pool through the salvage pathway. These and other findings suggest that a defect in guanine nucleotide biosynthesis evokes a reduction in the rate of general protein synthesis by impairing multiple steps of the process, disrupts the gene-specific reinitiation mechanism for translation of GCN4 mRNA and has far-reaching effects in cell biology and metabolism.