β-conglycinin, a soybean seed storage protein, is comprised of three different subunits, α, α', and β. Several candidates for the α subunit gene have been isolated, however, the structure of the α subunit gene has not been completely determined. Accordingly, it was also unknown which of the gene candidates are functionally active. Here, we have determined the nucleotide sequence and transcription start site of the α subunit gene, and compared structural components with those of the other subunits or other seed protein genes. The α subunit gene, which is located on a 7.6-kb EcoRI fragment, we composed of six exons that had the same organization as those for the α' subunit gene. Within a 400 bp upstream region of the transcription start site, four regions (designated as boxes I, II, III, and IV) were found to be conserved among the α, α', and other seed protein genes. Genomic Southern blot analysis of soybean varieties lacking the α subunit gene candidate indicated that the gene characterized in this paper actually encodes the α subunit and is functionally active. In addition, these experiments revealed the presence of an additional gene which is also responsible for the expression of the α subunit.