Pheromone-responsive conjugative plasmids are unique to the species Enterococcus faecalis. Many pheromone-responsive plasmids, including those frequently isolated from sites of infection, express a novel cytolysin that possesses both hemolytic and bacteriocin activities. Further, this cytolysin has been shown to be a toxin in several disease models. In the present study, nucleotide sequence determination, mutagenesis, and complementation analysis were used to determine the organization of the E. faecalis plasmid pAD1 cytolysin determinant. Four open reading frames are required for expression of the cytolysin precursor (cylLL, cylLS, cylM, and cylB). The inferred products of two of these open reading frames, CyILL and CyILS, constitute the cytolysin precursor and bear structural resemblance to posttranslationally modified bacteriocins termed lantibiotics. Similarities between the organization of the E. faecalis cytolysin determinant and expression units for lantibiotics exist, indicating that the E. faecalis cytolysin represents a new branch of this class and is the first known to possess toxin activity.