ABSTRACT Enterocin AS-48 production and immunity characters are encoded by 10 genes ( as - 48ABCC 1 DD 1 EFGH ) of the pMB2 plasmid from the Enterococcus faecalis S-48 strain. Among these, as - 48A , encoding the AS-48 peptide, and the as - 48BC genes constitute a cluster required for AS-48 biogenesis and full immunity. In this study, the levels of expression of this cluster have been altered by insertion and site-directed mutagenesis as well as by expression coupled to trans complementation. Phenotypic studies of the mutants have indicated cotranscription of the three genes and revealed that the inactivation of as - 48B prevents the production of AS-48, thus confirming its essentiality in AS-48 biogenesis. These studies have also supported the involvement of as - 48C in enterocin immunity. In addition, they established that the intergenic region between the as - 48A and as - 48B genes is decisive for AS-48 expression, since a 3-bp substitution, which should disrupt a potential 47-nucleotide complex secondary structure, resulted in a hypoproducing phenotype. Transcriptional analyses of the E. faecalis wild-type and mutant strains supports the possibility that the as - 48ABC genes are transcribed from the P A promoter located upstream of as - 48A . Moreover, analysis and bioinformatic predictions of RNA folding indicate that as - 48ABC mRNA is processed at the secondary structure located between as - 48A and as - 48B . Thus, synthesis of the AS-48 peptide appears to be controlled at the posttranscriptional level and is uncoupled from as - 48BC translation. This mechanism of genetic regulation has not been previously described for the regulation of bacteriocin expression in enterococci.