Lacticin 3147 is a two peptide lantibiotc (LtnA1 and LtnA2) that displays nanomolar activity against many Gram-positive bacteria. Lacticin 3147 may exert its antimicrobial effect by several mechanisms. Isothermal titration calorimetry experiments show that only LtnA1 binds to the peptidoglycan precursor lipid II, which could inhibit peptidoglycan biosynthesis. An experimentally supported model of the resulting complex that suggests the key binding partners are the C-terminus of LtnA1 and pyrophosphate of lipid II. A combination of in vivo and in vitro assays indicates that LtnA1 and LtnA2 can induce rapid membrane lysis without the need for lipid II binding. However, the presence of lipid II substantially increases the activity of lacticin 3147. Furthermore, studies with synthetic LtnA2 analogues containing either desmethyl- or oxa-lanthionine rings confirm that the precise geometry of these rings is essential for this synergistic activity.