The Helicobacter pylori vacuolating toxin VacA causes several effects on mammalian cells in vitro, including intracellular vacuolation, formation of pores in the plasma membrane and apoptosis. When added to cells, VacA becomes associated with detergent-resistant membranes, indicating that it binds preferentially to lipid rafts. In the present study, we have used atomic force microscopy to examine directly the association of VacA with lipid domains in supported lipid bilayers. VacA did not bind to lipid bilayers at pH 7.6. In contrast, at pH 4.0, VacA associated with the bilayers in the form of 26-nm oligomeric complexes. VacA bound to bilayers produced from either brain lipids or SM (sphingomyelin) plus cholesterol, each of which lacked detectable lipid domains. Bilayers composed of DOPC (dioleoylphosphatidylcholine), SM and cholesterol contained clearly visible raft-like domains, and VacA preferentially associated with these rafts. VacA bound poorly to raft-like domains in DOPC/SM bilayers, indicating that cholesterol is required for efficient association of VacA with lipid domains. When PS (phosphatidylserine), an anionic phospholipid that does not partition significantly into rafts, was added to the mixture of DOPC, SM and cholesterol, VacA was excluded from the rafts, indicating that it binds more avidly to PS than to the raft components. A typical plasma membrane exhibits pronounced lipid asymmetry, with SM enriched in the outer leaflet and PS in the inner leaflet. Therefore it is probable that the association of VacA with rafts in DOPC/SM/cholesterol bilayers represents a useful model for understanding the interactions of VacA with membranes in vivo.