Bacillus subtilis RB14 is a coproducer of the lipopeptide antibiotics iturin A and surfactin. Iturin A and surfactin have a similar structure consisting of a seven-amino-acid cyclic peptide, linked by either hydroxy or ester peptide linkage, respectively, to the fatty acid part. A 10-kb fragment responsible for the production of the lipopeptide antibiotics iturin A and surfactin in B. subtilis RB14 was minimized and the nucleotide sequence of the region essential for the synthesis of the lipopeptides was determined. A large open reading frame consisting of 224 amino acid residues was found. The gene, designated as lpa-14 (lipopeptide antibiotic production of RB14), showed high homology with sfp and psf-1 (regulators for surfactin production in B. subtilis and Bacillus pumilus, respectively) and an unknown open reading frame, orfX, in the upstream region of the peptide antibiotic gramicidin S biosynthesis operon of Bacillus brevis. The biosynthesis of surfactin and of iturin A was shown to be coregulated by the same gene, lpa-14. It is suggested that a common regulation system might exist among these genes for the production of peptide antibiotics in Bacillus species.