Amyloid β (Aβ) peptide contributes to Alzheimer's disease by a yet unidentified mechanism. In the brain tissue, Aβ occurs in various forms, including an undecapeptide Aβ25-35, which exerts a neurotoxic effect through the mitochondrial dysfunction and/or Ca2+-permeable pore formation in cell membranes. This work was aimed at the biophysical characterization of membrane binding and pore formation by Aβ25-35. Interaction of Aβ25-35 with anionic and zwitterionic membranes was analyzed by microelectrophoresis. In pore formation experiments, Aβ25-35 was incubated in aqueous buffer to form oligomers and added to Quin-2-loaded vesicles. Gradual increase in Quin-2 fluorescence was interpreted in terms of membrane pore formation by the peptide, Ca2+ influx, and binding to intravesicular Quin-2. The kinetics and magnitude of this process were used to evaluate the rate constant of pore formation, peptide-peptide association constants, and the oligomeric state of the pores. Decrease in membrane anionic charge and high ionic strength conditions significantly suppressed membrane binding and pore formation, indicating the importance of electrostatic interactions in these events. Circular dichroism spectroscopy showed that Aβ25-35 forms the most efficient pores in β-sheet conformation. The data are consistent with an oligo-oligomeric pore model composed of up to eight peptide units, each containing 6-8 monomers.