The production of the type I antimicrobial peptide (AMP) subtilin by Bacillus subtilis is regulated in a cell-density-dependent manner [Kleerebezem M, de Vos WM, Kuipers OP. The lantibiotics nisin and subtilin act as extracellular regulators of their own biosynthesis. In: Dunny GM, Winans SC, editors. Cell-cell signaling in bacteria. Washington, D.C., USA: ASM Press; 1999. p. 159-74; Stein T, Borchert S, Kiesau P, Heinzmann S, Kloss S, Klein C, Helfrich M, Entian KD. Dual control of subtilin biosynthesis and immunity in Bacillus subtilis. Mol Microbiol 2002;44:403-16; Stein T, Heinzmann S, Kiesau P, Himmel B, Entian KD. The spa-box for transcriptional activation of subtilin biosynthesis and immunity in Bacillus subtilis. Mol Microbiol 2003;47:1627-36]. Three subtilin-responsive promoter elements within the spaBTCSIFEGRK are controlled by the specific cis-acting sequence element called the spa-box, which represents the binding site of the subtilin regulator SpaR [Stein T, Heinzmann S, Kiesau P, Himmel B, Entian KD. The spa-box for transcriptional activation of subtilin biosynthesis and immunity in Bacillus subtilis. Mol Microbiol 2003;47:1627-36]. Here, we describe the functional characterization of the spaB, spaS and spaI promoters by transcriptional fusion with a promoterless beta-glucuronidase encoding gusA gene. Within these gusA fusion constructs, transcription initiation start sites of the spaS and spaI promoters were mapped to be located downstream of the spa-box, which is in contrast to previous reports [Banerjee S, Hansen JN. Structure and expression of a gene encoding the precursor of subtilin, a small protein antibiotic. J Biol Chem 1988;263:9508-14; Stein T, Heinzmann S, Kiesau P, Himmel B, Entian KD. The spa-box for transcriptional activation of subtilin biosynthesis and immunity in Bacillus subtilis. Mol Microbiol 2003;47:1627-36]. Nevertheless, all spa-promoters displayed typical cell-density-dependent activity in a subtilin-producing strain B. subtilis ATCC6633. Moreover, analysis of beta-glucuronidase activities in a spaB mutant of B. subtilis ATCC6633 and a derivative of strain 168 that harbors the spaRK genes integrated in the chromosomal amyE locus, confirmed that these promoters are activated by subtilin-triggered, SpaRK-mediated, quorum-sensing control. Quantitative analysis showed that the spaS promoter strength at a given subtilin concentration appeared to be approximately five-fold higher than the spaB promoter, which in turn is approximately two-fold higher than the spaI promoter. Finally, it is shown that the elementary components involved in subtilin-mediated regulation are the two-component system, SpaRK, and a spa-box containing promoter.