Genetic reversion of HIS4 initiator codon mutations in yeast has identified three unlinked genes, sui1, sui2, and SUI3 (suppressors of initiator codon mutations), which when mutated confer the ability to initiate translation at HIS4 despite the absence of an AUG start codon. We have previously demonstrated that the SUI3 gene encodes the beta subunit of the eukaryotic initiation factor 2 (eIF-2) and that mutations at a Zn(II) finger motif of SUI3 alter the start site selection process in yeast. In this report, molecular and biochemical characterizations show that the sui2 suppressor gene encodes the alpha subunit of eIF-2. The amino acid sequence of sui2 is 58% homologous to that encoded by the cDNA of the human eIF-2 alpha. Mutations in the sui2 suppressor alleles occur in the amino-terminal portion of the protein and change amino acids that are identical at the same relative position in the yeast and human proteins. Protein sequence analysis shows that a sui2 mutant yeast strain allows initiation at a UUG codon in the absence of an AUG codon at HIS4. These data further suggest that eIF-2 is an important component of the preinitiation complex that mediates ribosomal recognition of a start codon during the scanning process.