Microstamping on an activated polymer surface (MAPS) is a methodology that enables biomolecules to be patterned on polymers with micrometer spatial resolution. MAPS combines homogeneous surface derivatization of a polymer to introduce a reactive functional group followed by reactive microcontact printing (mu CP) of a biological ligand of interest, linked to an appropriate reactive group. We demonstrate here that polyethylene, polystyrene, poly(methyl methacrylate), and poly(ethylene terephthalate) films can be successfully modified to introduce COOH groups on their surfaces, which can be subsequently patterned by reactive mu CP of amine-terminated biotin after derivatization. of the COOH groups with pentafluorophenol. X-ray photoelectron spectroscopy and time-of-flight secondary ion mass spectrometry (TOF-SIMS) confirmed the chemistry of MAPS at each stage of the derivatization of the polymer surfaces and reactive mu CP of biotin. Micropatterned biotin surfaces fabricated by MAPS were patterned with streptavidin by exploiting molecular recognition between biotin and streptavidin. The formation of streptavidin patterns was examined by fluorescence microscopy of Alexa488-labeled streptavidin and by TOF-SIMS imaging of N-15-labeled recombinant streptavidin, bound to biotin patterns. The contrast in the streptavidin micropatterns was optimized by examining the effect of blocking agents and streptavidin incubation time. Maximum contrast was obtained for binding of 0.1 muM streptavidin from a buffer containing 0.02% (v/v) Tween 20 detergent for an incubation time of 1 min.