Lacticin 3147 is a membrane-active, two-component lantibiotic produced by Lactococcus lactis ssp. lactis DPC3147. In this study, the promoters of the lacticin 3147 gene cluster were mapped to the intergenic region between ItnR and ItnA(1) (the genes encoding the regulatory protein LtnR and the first structural gene, LtnA1), and Northern analyses revealed that the biosynthetic and immunity genes are divergently transcribed in two operons, ItnA(1)A(2)M(1)TM(2)D and ItnRIFE respectively. Although the promoter controlling biosynthesis (P-bac) appears to be constitutive, characterization of a downstream beta -galactosidase (beta -gal) fusion beyond an intragenic stem-loop structure in ItnM(1) confirmed that this putative transcriptional attenuator allows limited readthrough to the downstream biosynthetic genes, thus maintaining the correct stoichiometry between structural peptides and biosynthetic machinery. The promoter of the ItnRIFE operon (P-imm) was shown to be regulated by the transcriptional repressor LtnR. A mutant with a truncated ItnR gene exhibited a hyperimmune phenotype, whereas overexpression of ItnR resulted in cells with increased sensitivity to lacticin 3147. Gel mobility shift analysis indicated that LtnR binds to the P-imm promoter region, and fusion of this promoter to the beta -gal gene of pAK80 revealed that expression from P-imm is significantly reduced in the presence of LtnR. Thus, we have demonstrated that lacticin 3147 uses a regulatory mechanism not previously identified in lantibiotic systems.