To study the regulatory mechanism of expression of the plant gene, soybean glycinin genes were isolated. A full length cDNA clone for glycinin subunit A₂B_(1a) was isolated by screening a cDNA expression library and determined its nucleotide sequence. Gemonic DNA clone of glycinin was isolated by plaque hybridization of a genomic library with the cDNA probe. It demonstrated that the genomic clone λG47 contained two glycinin genes, Gy1 and Gy2, in the same orientation with a intergenic region of 2.7 kbp in size. The structure of the glycinin gene, Gy2 was determined by nucleotide sequencing. The Gy2 gene consists of 4 exons and 3 introns, spanning over 2,863 bp and encodes 485 amino acids. Gel retardation assay with the nuclear extract prepared from developing seeds demonstrated that nuclea proteins bind to DNA fragments up to -256 bp from the transcription initiation site which contain the putative regulatory elements. The non-radioactive probes compete off the nuclear factors bound to the radioactive probes, demonstrating their sequencespecific interaction. DNase I footprint analysis showed that the region around -63 was protected from DNase I digestion, which contains a SV 40 enhancer-like sequence. The regulatory effect of these DNA elements was tested with Gy2 promoter deletion mutants fused to the GUS reporter gene. Transient assay with soybean seed protoplasts revealed that the Gy2 promoter regulates GUS expression in biphase. These results suggested the presence of negative regulatory sequences or repressors bound to these domains. Combination of these negative and positive regulatory elements could maintain tissue- and development-specific gene expression pattern of the glycinin gene.