The heterotrimeric eukaryotic initiation factor (eIF) 2 binds the initiator methionyl-tRNA in a GTP-dependent mode and delivers it to the 40S ribosomal subunit. In the present study, we have identified amino acid residues in eIF2β required for binding to eIF2γ in yeast. Alteration of six residues in the central region of eIF2β abolished this interaction, as determined by GST-pull down and two-hybrid assays, and leads to cell lethality. Substitution of 131Tyr and 132Ser by alanine residues (131YS), although abolishing the binding to eIF2γ in these assays, resulted in a functional but defective protein in vivo, imparting a temperature-sensitive growth phenotype to cells. A dramatically weakened association of this mutant protein with eIF2γ in vivo was shown by co-immunoprecipitation. The 131YS mutation in eIF2β allows translation to initiate at non-AUG codons, as defined by the ability of cells carrying an initiator codon mutation in the HIS4 mRNA to grow in the absence of histidine. The combination of this mutation with the 264Ser→Tyr alteration, a previously isolated suppressor of initiator codon mutations which has been shown to increase the spontaneous GTP hydrolysis in the ternary complex, caused a recessive lethality, suggesting additive defects. Thus the impaired interaction of these two subunits represents a novel type of defect in eIF2 function, providing in vivo evidence that the strength of interaction between eIF2β and eIF2γ defines the correct usage of the AUG codon for translation initiation.