Nisin is a model system for lantibiotics, a class of peptides displaying antimicrobial activity against various Gram-positive bacteria. After ribosomal synthesis, the precursor peptide is modified in two steps, of which the last one involves consecutive cyclization reactions mediated by the cyclase NisC. Here, we present a detailed in vitro study of the interaction between NisC and the nisin precursor peptide. Our results unravel a specific interaction of NisC with the leader peptide independent of the maturation state. Furthermore, mutagenesis studies identified a specific binding sequence within the leader. Two amino acids (F-18 and L-16) within the highly conserved -FNLD- box of class I lantibiotics are essential for binding. They represent a potential general binding motif between leader peptides of a group of lantibiotics with their cyclase family. In summary, these in vitro data provide a new perception on the complexity of the lantibiotic modification machineries.