A new and sensitive method for quantifying high affinity binding of proteins land potentially other ligands) to the surfaces of phospholipid bilayered vesicles is demonstrated A planar, high-density streptavidin monolayer is first immobilized onto a surface plasmon resonance (SPR) sensor slide containing a mixed monolayer of biotin-terminated and hydroxyl-terminated poly(ethylene oxide) alkylthiolates tethered to the gold surface. Phospholipid vesicles containing 0.3% biotin-functionalized headgroups are then bound to this streptavidin monolayer to make a high-density, planar layer of intact vesicles. The absolute amount of protein binding to the vesicle layer can be monitored by SPR in real time to extract equilibrium and kinetic information under flowing solutions. Coupled with a catalytic assay to monitor the solution-phase concentration of enzyme, the value of the dissociation equilibrium constant for the complex of cobra venom phospholipase A(2) (PLA(2)) bound to phosphatidylcholine vesicles was determined to be 6 +/- 2 x 10(-7) M. In principle, the method could be extended to determine dissociation constants as low as 10(-10) M. The vesicles bind a maximum of 1 PLA(2) per 74 +/- 16 outer leaflet phospholipids. The method provides a practical solution to a number of problems encountered with previous methods to quantify tight interfacial binding of proteins to vesicles. Spectral probes attached to enzymes or vesicles, which may perturb interfacial binding, are not required. Furthermore, surface-supported vesicles offer improved sensitivity over planar bilayers and are physiologically more relevant.