The self-assembly of streptavidin onto biotinylated alkylthiolate monolayers on gold has served as an important model system for protein immobilization at surfaces. Here, we report a detailed study of the surface composition and structure of mixed self-assembled monolayers (SAMs) containing biotinylated and diluent alkylthiolates and their use to specifically immobilize streptavidin. X-ray photoelectron spectroscopy (XPS), angle-resolved XPS (ARXPS), near-edge X-ray absorption fine structure (NEXAFS), and surface plasmon resonance (SPR) have been used to characterize the films produced on gold from a range of binary mixtures of a biotinylated alkylthiol (BAT) and either a Cls methyl-terminated thiol (mercaptohexadecane, MHD) or a C-11-oligo(ethylene glycol)-terminated (OEG) thiol in ethanol. The correlation between the solution mole fraction of BAT and its surface mole fraction (chi (BAT,sur)) indicates that it adsorbs similar to4-fold faster than OEG but slightly slower than MHD. ARXPS analysis demonstrates that the biotin terminus of the BAT is exposed at the surface of mixed monolayers with chi (BAT,sur) < 0.5 but is randomly distributed through BAT-rich films. Thus, the OEG diluent not only adds nonfouling properties but induces an improved concentration of biotin at the surface and reduces the exposure of the methylene segments of BAT. NEXAFS characterization demonstrates that pure OEG and mixed BAT/OEG SAMs do not show significant anisotropy in C-C bond orientation, in contrast to MHD and mixed BAT/MHD SAMs, whose aliphatic segments exhibit pseudo-crystalline packing. SPR measurements of streptavidin binding to and competitive dissociation from the different mixed SAMs indicate that streptavidin binds both specifically and nonspecifically to the BAT/MHD SAMs but purely specifically to BAT/OEG SAMs with chi (BAT,sur) < 0.5 For BAT/OECT mixtures with chi (BAT,sur) = 0.1-0.5, specifically bound streptavidin coverages of similar to 80% of the C(2,2,2) two-dimensional streptavidin crystalline density (similar to 280 ng/cm(2)) can be reproducibly achieved. These composite results clarify the relationship between the specificity of streptavidin recognition and the surface architecture and properties of the mixed SAMs.