Some 8.8 kb of the Lactobacillus sake plasmid pCIM1 was sequenced, revealing eight tightly clustered open reading frames (ORFs) downstream from lasA, which encodes pre-lactocin S. Transcription analyses demonstrated that the genes are expressed as an operon, with transcription initiating upstream of lasA and terminating immediately 3′ to the ninth ORF · lasA is also represented by two small RNAs (RNAI and RNAII) which differ in size by approximately 90 nucleotides, and primer extension experiments demonstrated a corresponding difference in the 5′ termini. A palindromie sequence constitutes the 3′ terminus of both RNAI and RNAII, and we propose that this sequence has a dual regulatory function in controlling the expression of las operon, acting both as a barrier to 3′–5′ exonuclease degradation of the lasA-specific transcript(s), and as a “leaky” transcriptional terminator which limits the expression of down-stream genes. Three of the genes in the las operon have identifiable counterparts in other lantibiotic systems: lasM is likely to be involved in prepeptide modification, lasT, which encodes an ATP-dependent transport protein, is probably involved in the secretion of lactocin S, while lasP specifies a subtilisin-type serine protease which may be the lactocin S leader peptidase. Insertional mutation of either lasT or lasM by the resident transposable element IS1163 abolishes lactocin S production. The remaining five ORFs in the las operon are apparently unique, and their significance with respect to the lactocin S phenotype is presently not known.