Surface pressure measurements, external reflection-Fourier transform infrared spectroscopy, and neutron reflectivity have been used to investigate the lipid-binding behavior of three antimicrobial peptides: melittin, magainin II, and cecropin P1. As expected, all three cationic peptides were shown to interact more strongly with the anionic lipid, 1,2 dihexadecanoyl-sn-glycerol-3-(phosphor-rac-(1-glycerol)) (DPPG), compared to the zwitterionic lipid, 1,2 dihexadecanoyl-sn-glycerol-3-phosphocholine (DPPC). All three peptides have been shown to penetrate DPPC lipid layers by surface pressure, and this was confirmed for the melittin-DPPC interaction by neutron reflectivity measurements. Adsorption of peptide was, however, minimal, with a maximum of 0.4 mg m(-2) seen for melittin adsorption compared to 2.1 mg m(-2) for adsorption to DPPG (from 0.7 microM solution). The mode of binding to DPPG was shown to depend on the distribution of basic residues within the peptide alpha-helix, although in all cases adsorption below the lipid layer was shown to dominate over insertion within the layer. Melittin adsorption to DPPG altered the lipid layer structure observed through changes in the external reflection-Fourier transform infrared lipid spectra and neutron reflectivity. This lipid disruption was not observed for magainin or cecropin. In addition, melittin binding to both lipids was shown to be 50% greater than for either magainin or cecropin. Adsorption to the bare air-water interface was also investigated and surface activity followed the trend melittin>magainin>cecropin. External reflection-Fourier transform infrared amide spectra revealed that melittin adopted a helical structure only in the presence of lipid, whereas magainin and cecropin adopted helical structure also at an air-water interface. This behavior has been related to the different charge distributions on the peptide amino acid sequences.