The aim of this work was to create patterned surfaces for localized and specific biochemical recognition. For this purpose, we have developed a protocol for orthogonal and material-selective surface modifications of microfabricated patterned surfaces composed of SiO2 areas (100 mu m diameter) surrounded by Au. The SiO2 spots were chemically modified by a sequence of reactions (silanization using an amine-terminated silane (APTES), followed by amine coupling of a biotin analogue and biospecific recognition) to achieve efficient immobilization of streptavidin in a functional form. The surrounding Au was rendered inert to protein adsorption by modification by HS(CH2)(10)CONH-(CH2)(2)(OCH2CH2)(7)OH (thiol-OEG). The surface modification protocol was developed by testing separately homogeneous SiO2 and Au surfaces, to obtain the two following results: (i) SiO2 surfaces which allowed the grafting of streptavidin, and subsequent immobilization of biotinylated antibodies, and (ii) Au surfaces showing almost no affinity for the same streptavidin and antibody solutions. The surface interactions were monitored by quartz crystal microbalance with dissipation monitoring (QCM-D), and chemical analyses were performed by polarization modulation-reflexion absorption infrared spectroscopy (PM-RAIRS) and X-ray photoelectron spectroscopy (XPS) to assess the validity of the initial orthogonal assembly of APTES and thiol-OEG. Eventually, microscopy imaging of the modified Au/SiO2 patterned substrates validated the specific binding of streptavidin on the SiO2/APTES areas, as well as the subsequent binding of biotinylated anti-rIgG and further detection of fluorescent rIgG on the functionalized SiO2 areas. These results demonstrate a successful protocol for the preparation of patterned biofunctional surfaces, based on microfabricated Au/SiO2 templates and supported by careful surface analysis. The strong immobilization of the biomolecules resulting from the described protocol is advantageous in particular for micropatterned substrates for cell-surface interactions.