Drop-shape analysis was used to study the binding of streptavidin to biotin at the interface between water and a pendant chloroform droplet. Polyethylene oxide molecules were synthesized with a hydrophobic tail at one end of the molecule and a hydroxyl or biotin group at the other end. The interfacial tension of the water/chloroforrn interface was measured before and after addition of these amphiphiles to the chloroform phase and before and after addition of streptavidin to the aqueous phase. The hydroxyl-terminated amphiphiles eliminate nonspecific adsorption of the streptavidin to the interface, while streptavidin binds irreversibly to the biotin-terminated molecules. Molecular interactions within this bound layer were studied by measuring changes in the interfacial pressure as the layer is contracted and expanded by changing the volume of the chloroform droplet. A picture of the interfacial structure was obtained from quantitative comparisons between the experimental results and a molecular theory of protein binding to tethered ligands. These comparisons suggest that protein binding is controlled by the extension of-the PEO tethers away from the interface.