Detailed insight into the interplay between antimicrobial peptides and biological membranes is fundamental to our understanding of the mechanism of bacterial ion channels and the action of these in biological host-defense systems. To explore this interplay, we have studied the incorporation, membrane-bound structure, and conformation of the antimicrobial peptide alamethicin in lipid bilayers using a combination of H-1 liquid-state NMR spectroscopy and molecular dynamics (MD) simulations. On the basis of experimental NMR data, we evaluate simple in-plane and transmembrane incorporation models as well as pore formation for alamethicin in DMPC/DHPC (1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine/1,2-dihexanoyl-sn-glyc ero-3-phosphatidyicholine) bicelles. Peptide-lipid nuclear Overhauser effect (NOE) and paramagnetic relaxation enhancement (PRE) data support a transmembrane configuration of the peptide in the bilayers, but they also reveal that the system cannot be described by a single simple conformational model because there is a very high degree of dynamics and heterogeneity in the three-component system. To explore the origin of this heterogeneity and dynamics, we have compared the NOE and PRE data with MD simulations of an ensemble of alamethicin peptides in a DMPC bilayer. From all-atom MD simulations, the contacts between peptide, lipid, and water protons are quantified over a time interval up to 95 ns. The MD simulations provide a statistical base that reflects our NMR data and even can explain some initially surprising NMR results concerning specific interactions between alamethicin and the lipids.