Electrochemical impedance spectroscopy (EIS), atomic force microscopy (AFM), and photon polarization modulation infrared reflection absorption spectroscopy (PM-IRRAS) were employed to investigate the formation of alamethicin pores in negatively charged bilayers composed of a mixture of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and egg PG floating at gold (111) electrode surfaces modified by self-assembled monolayers of 1-thio-beta-D-glucose (beta-Tg). The EIS data showed that the presence of alamethicin decreases the membrane resistivity by about 1 order of magnitude. PM-IRRAS measurements provided information about the tilt angles of peptide helical axis with respect to the bilayer normal. The small tilt angles obtained for the peptide helical axis prove that the alamethicin molecules were inserted into the DMPC/egg-PG membranes. The tilt angles decreased when negative potentials were applied, which correlates with the observed decrease in membrane resistivity, indicating that ion pore formation is assisted by the transmembrane potential. Molecular resolution AFM images provided visual evidence that alamethicin molecules aggregate forming hexagonal porous 2D lattices with periodicities of 2.0 +/- 0.2 nm. The pore formation by alamethicin in the negatively charged membrane was compared with the interaction of this peptide with a bilayer formed by zwitterionic lipids. The comparison of these results showed that alamethicin preferentially forms ion translocating pores in negatively charged phospholipid membranes.