An 85 by DNA fragment, the nucleotide sequence of which had 84% homology with the sequence for the promoter, ribosome binding site and NH₂-terminal five amino acids of the Bacillus amyloliquefaciens α-Amylase gene, was chemically synthesized. In order to analyze the promoter activity of a Bacillus subtilis α-Amylase secretion vector, the fragment was inserted between the promoter and signal peptidecoding region of Bacillus subtilis α-Amylase gene. Both promoters, tandemly repeated, functioned in transcribing the B. subtilis α-Amylase signal peptide-coding region followed by the Escherichia coli β-lactamase structural gene. The transcrip-tion initiation sites were determined by the primer extension method. The extracellular production of β-lactamase was stimulated by two promoters as compared with that by the plasmids containing either promoter region alone. The change of two amino acids in the NH₂-terminal region of the B. subtilis α-Amylase signal peptide had no effect on the secretion of β-lactamase from B. subtilis cells.