We have tested the ability of Amphotericin B to form ion channels/defects in osmotically stressed large unilamellar vesicles (LUV) using pyranine fluorescence detected ion/H+ exchange. We found that sterol-free LUV exhibit greatly increased sensitivity to AmB channel formation in the soluble oligomer state (> 0.5 microM) under modestly hypoosmotic conditions (< 100 delta mosM). These vesicles are completely insensitive under isoosmotic conditions. The related antibiotics, Amphotericin B methyl ester and Nystatin showed almost no activity under hypoosmotic conditions in the absence of sterol. This difference may be attributable to differences in solution oligomeric states. Experiments with KCl and CaCl2 internal buffers demonstrate that these sterol-free AmB membrane disruptions are highly selective for monovalent cations (K+) over anions (Cl-), ruling out massive lysis or unselective membrane defects caused by osmotic pressure. Thus, AmB seems to be acting as a 'molecular harpoon', an expression coined to describe substances which can selectively target osmotically stressed, strained or highly curved membranes. These results may provide a rationale for AmB's reported anti-HIV activity and reported activity against sterol-free small unilamellar vesicles (highly curved membranes) as well as the reduced activity of liposomal drug delivery systems toward cholesterol-containing and sterol-free membranes (fewer soluble oligomers).