The interaction of pore-forming streptolysin O (SLO) with biomimetic lipid membranes has been studied by electrochemical methods. Phosphatidylcholine lipid vesicles were deposited onto gold electrodes modified with supporting layers of hexyl thioctate (HT) or thioctic acid tri(ethylene glycol) ester (TA-TEGE), and integrity and permeability of the resulting membranes were characterized by cyclic voltammetry and impedance spectroscopy. Both positively and negatively charged electrochemical probes, potassium ferrocyanide, hexaammineruthenium(III) chloride, and ferrocene carboxylic acid (FCA), were employed to evaluate their suitability to probe the membrane permeability properties, with FCA exhibiting ideal behavior and thus employed throughout the work. Fusion of vesicles incubated with SLO on the electrodes yielded membranes that showed a distinctive response pattern for FCA as a function of SLO concentration. A direct dependence of both the currents and peak separation of FCA in the cyclic voltammograms was observed over a concentration range of 0-10 hemolytic units (HU)/mu L of the toxin. The interaction of SLO with preformed supported lipid membranes was also investigated, and much lower response was observed, suggesting a different extent of membrane-toxin interactions on such an interface. Nonionic surfactant Triton was found to disrupt the vesicle structure but could not completely remove a preformed membrane to fully restore the electrode response. The information reported here offers some unique insight into toxin-surface interactions on a hybrid membrane, facilitating the development of electrochemically based sensing platforms for detecting trace amounts of bacterial toxins via the perforation process.