In a previous report we described the selection of potent, beta-sheet pore-forming peptides from a combinatorial library designed to mimic membrane-spanning beta-hairpins (Rausch, J. M., Marks, J. R., and Wimley, W. C. (2005) Proc. Natl. Acad. Sci. U.S.A. 102, 10511-10515). Here, we characterize their mechanism of action and compare the structure-function relationships in lipid vesicles to their activity in biological membranes. The pore-forming peptides bind to membrane interfaces and self-assemble into beta-sheets that cause a transient burst of graded leakage across the bilayers. Despite the continued presence of the structured peptides in the bilayer, at most peptide concentrations leakage is incomplete and ceases quickly after peptide addition with a deactivation half-time of several minutes. Molecules up to 3,000 Da escape from the transient pores, but much larger molecules do not. Fluorescence spectroscopy and quenching showed that the peptides reside mainly on the bilayer surface and are partially exposed to water, rather than in a membrane-spanning state. The "carpet" or "sinking raft" model of peptide pore formation offers a viable explanation for our observations and suggests that the selected pore-formers function with a mechanism that is similar to the natural pore-forming antimicrobial peptides. We therefore also characterized the antimicrobial and cytotoxic activity of these peptides. All peptides studied, including non-pore-formers, had sterilizing antimicrobial activity against at least some microbes, and most have low activity against mammalian cell membranes. Thus, the structure-function relationships that were apparent in the vesicle systems are similar to, but do not correlate completely with, the activity of the same peptides in biological membranes. However, of the peptides tested, only the pore-formers selected in the high-throughput screen have potent, broad-spectrum sterilizing activity against Gram-positive and Gram-negative bacteria as well as against fungi, while having only small lytic effects on human cells.