A wide variety of naturally expressed anti-microbial peptides (AMPs) have been discovered in plants, animals, and humans. AMPs are remarkably for their general ability to halt growth of both Gram negative and Gram positive bacteria. Cationic AMPs bind to negatively charged bacterial cell membranes and evidently degrade their barrier function, eventually leading to the halting of growth and cell death. The relative inabilit of bacteria to resist this general mode of action makes AMPs and their mimics interesting drug candidates against antibiotic-resistant strains. Almost all mechanistic studies thus far have focused either on synthetic lipid vesicles in vitro or on long-time, bulk effects of AMPs on bacteria. The detailed mechanisms of AMP attack on bacterial membranes are not well understood. This work develops novel fluorescence microscopy assays in order to directly observe the attack of AMPs on bacterial membranes for individual cells in real time. On attack of ?-helical peptides on E. coli, we observe such events as: binding to and diffusion within the outer membrane (OM), translocation across the OM, permeabilization of the OM to periplasmic GFP, abrupt cell shrinkage and the halting of cell growth, permeabilization of the cytoplasmic membrane (CM) to the dye Sytox Green, and the onset of oxidative damage within the cytoplasm. We plan to extend our work from ?-helical AMPs to defensins, the other important class of human AMPs. In addition to studies of the model species E. coli and B. subtilis, we will observe AMP effects on non-pathogenic strains of P. aeruginosa and S. aureus. We will initiate studies of AMP activity against bacteria living in model biofilms. Our results will test the relevance of previous studies on synthetic lipid bilayers to growth-halting mechanisms in real bacteria. It is critical that we develop new means to kill drug-resistant pathogens. By dissecting the steps by which natural AMPs kill bacteria, we will provide new design criteria for synthetic mimics of AMPs that may prove clinically useful. The novel methods developed here will be widely applicable to mechanistic studies of natural AMPs, synthetic copolymers designed to mimic AMPS, and synthetic drugs.