Translation initiation is down-regulated in eukaryotes by phosphorylation of the alpha-subunit of eIF2 (eukaryotic initiation factor 2), which inhibits its guanine nucleotide exchange factor, eIF2B. The N-terminal S1 domain of phosphorylated eIF2alpha interacts with a subcomplex of eIF2B formed by the three regulatory subunits alpha/GCN3, beta/GCD7, and delta/GCD2, blocking the GDP-GTP exchange activity of the catalytic epsilon-subunit of eIF2B. These regulatory subunits have related sequences and have sequences in common with many archaeal proteins, some of which are involved in methionine salvage and CO(2) fixation. Our sequence analyses however predicted that members of one phylogenetically distinct and coherent group of these archaeal proteins [designated aIF2Bs (archaeal initiation factor 2Bs)] are functional homologs of the alpha, beta, and delta subunits of eIF2B. Three of these proteins, from different archaea, have been shown to bind in vitro to the alpha-subunit of the archaeal aIF2 from the cognate archaeon. In one case, the aIF2B protein was shown further to bind to the S1 domain of the alpha-subunit of yeast eIF2 in vitro and to interact with eIF2Balpha/GCN3 in vivo in yeast. The aIF2B-eIF2alpha interaction was however independent of eIF2alpha phosphorylation. Mass spectrometry has identified several proteins that co-purify with aIF2B from Thermococcus kodakaraensis, and these include aIF2alpha, a sugar-phosphate nucleotidyltransferase with sequence similarity to eIF2Bvarepsilon, and several large-subunit (50S) ribosomal proteins. Based on this evidence that aIF2B has functions in common with eIF2B, the crystal structure established for an aIF2B was used to construct a model of the eIF2B regulatory subcomplex. In this model, the evolutionarily conserved regions and sites of regulatory mutations in the three eIF2B subunits in yeast are juxtaposed in one continuous binding surface for phosphorylated eIF2alpha.