The genetic determinants for production and immunity to the two-component lantibiotic lacticin 3147 are encoded by a 12.6 kb region of the plasmid pMRC01. this region contains ten genes arranged in two divergent clusters; these include the structural genes and a number of genes whose products show significant similarity to proteins involved in the biosynthesis of other lantibiotics, Using a strategy of deletion and mutational analysis, the effect of disruption of a number of these genes was investigated. Inactivation of either of the structural genes, ItnA1 or ItnA2, resulted in mutants that were incapable of producing active lacticin 3147; however, the combination of the cell-free supernatant from both mutants resulted in a restoration of bacteriocin activity, confirming that processing and export of the structural peptides can occur independently. An unusual feature of the lacticin 3147 gene cluster is the presence of two lanM homologues, whose gene products are proposed to be involved in the dehydration and thioether-forming reactions which result in lanthionine bridge formation. Mutants created in the ItnM1 and ItnM2 genes were also incapable of lantibiotic production, confirming an essential role for these enzymes in the lacticin 3147 biosynthetic pathway and supporting the assertion that these proteins are modification enzymes. Interestingly, addition of purified LtnA1, but not purified LtnA2, to the cell-free supernatant of the ItnM1 mutant restored bacteriocin activity; in contrast, only purified LtnA2 could complement the cell-free supernatant of the ItnM2 mutant. Creation of a number of double mutants supported these findings, and confirmed that LtnM1 is required to produce mature LtnA1, while LtnM2 is required to produce mature LtnA2.