Host Defence Peptides (HDPs), generally termed defensins, are major effector molecules of innate immune systems. Among the HDPs of the human alpha- and beta-defensins, hBD3 is the most cationic (+11); it retains activity at elevated salt concentrations which is relevant for the control of staphylococci on the skin and mucosal surfaces. Staphylococci are able to sense HDPs and their antibiotic effects through the GraRS two-component regulatory system and, in a coordinated respose, reduce the the negative charge of the cell evelope. We studied hBD3, using S. aureus SG511 as test strain, to elcudiate the killing mechanism of defensins which is generally believed to depend on membrane permeabilization. We found little evidence that disruption of membrane barrier functions significantly contributes to killing. Analysis of transcriptional responses to hBD3 treatment and whole cell assays suggest that the functionality of multi-enzyme machines is impaired, e.g. transporters, the respiratory chain and, of particular relevance, cell wall biosynthesis. In vitro testing of individual cell wall biosynthesis reaction suggests that Lipid II polymerization on the outside of the membrane is inhibited. Obviously, "membrane-active", amphiphilic host defence peptides disrupt membrane associated biosynthetic processes rather than the lipid bilayer itself.