Antimicrobial peptides (AMPs) are natural bactericidals and immunomodulators, and their action involves association to cell membranes and their perturbation. In vitro, they are selective for bacteria versus eukaryotic cells. Although AMPs are promising compounds to fight drug-resistant bacteria, many unanswered questions hinder their therapeutic application: is their main role bactericidal or immunomodulatory? What are the structural determinants of selectivity, other than their cationic charge? Is cell selectivity real or an artifact of the assay conditions?[1] By combining spectroscopic experiments on liposomes and cells and molecular dynamics simulations, we characterized how AMP activity and selectivity are regulated by multiple thermodynamic equilibria: membrane binding, aggregation, conformational transitions and orientation in the bilayer.[2] By measuring peptide association to bacteria and erythrocytes, we found that high coverage of the cell surface is needed to cause its death/lysis, showing that the carpet mechanism is relevant in real cells. Due to the binding equilibrium, the total peptide concentration in solution necessary to reach this threshold never decreases below micromolar values, even at low cell densities. These data indicate that immunomodulation might prevail where these concentrations are not reached, and provide a new insight in the cell density dependence of selectivity. In addition, we showed that peptide folding or aggregation in solution are ways to increase peptide selectivity, by shielding the hydrophobic residues and reducing the hydrophobic-driving force for binding to the neutral surface of eukaryotes. Overall, our studies indicate that characterization and modulation of the thermodynamic equilibria involved in peptide behavior are a valuable approach to understand AMP function and to improve their properties.1) NatChemBiol 2013 9:761; BiochimBiophysActa 2009 1788:1687.2) BiochimBiophysActa 2015 1848:581; ACS ChemBiol 2014 9:2003; JPeptSci. 2013 19:758; BiochimBiophysActa 2013 1828:1013; Biochemistry 2012 51:10124; CellMolLifeSci 2011 68:2281; BiophysJ 2010 99:1791.