The lantibiotic nisin is produced by several strains of Lactococcus lactis subsp. lactis. The chromosomally located gene cluster nisABTCIPRKFEG is required for biosynthesis, development of immunity, and regulation of gene expression. Inframe deletions in the nisB and nisT genes, and disruption of nisC by plasmid integration, eliminated nisin production and resulted in a strongly reduced level of immunity of the strains. The transcription of two nisin operons was inactivated in these mutant strains, but could be restored by addition of small amounts of nisin to growing cultures. The immunity levels of the mutants were also raised by adding nisin to growing cultures, albeit not to wild-type level. A strain with an in-frame deletion in the nisI gene was still able to produce active nisin, but the production and immunity levels were markedly lower. By measuring immunity levels of the knock-out strains and determining mRNA levels, it is concluded that NisI has an important function for nisin immunity and must cooperate with nisFEG-encoded proteins to provide a high level of immunity. Maximal immunity could not be obtained in the mutant strains, probably because the wild-type transcription levels from nisA and nisF promoters are not reached when essential nis genes are disrupted. Using Southern hybridization with a consensus promoter probe, no other DNA sequences similar to the nisA and nisF promoters could be detected, indicating that these two elements are probably the only ones in the chromosome regulated by nisin and are thus the only ones involved in the regulation of producer immunity.