The lantibiotic nisin is produced by Lactococcus lactis. In the biosynthesis of nisin, the enzyme NisB dehydrates nisin precursor, and the enzyme NisC is needed for lanthionine formation. In this study, the nisA gene encoding the nisin precursor, and the genes nisB and nisC of the lantibiotic modification machinery were expressed together in vitro by the Rapid Translation System (RTS). Analysis of the RTS mixture showed that fully modified nisin precursor was formed. By treating the mixture with trypsin, active nisin was obtained. However, no nisin could be detected in the mixture without zinc supplementation, explained by the fact that NisC requires zinc for its function. The results revealed that the modification of nisin precursor, which is supposed to occur at the inner side of the membrane by an enzyme complex consisting of NisB, NisC, and the transporter NisT, can take place without membrane association and without NisT. This in vitro production system for nisin opens up the possibility to produce nisin variants that cannot be producedin vivo. Moreover, the system is a promising tool for utilizing the NisB and NisC enzymes for incorporation of thioether rings into medical peptides and hormones for increased stability.