We report here molecular characterization of a new method derived from reactive microcontact printing microstamping on an activated polymer surface (MAPS)-which enables biological ligands and proteins to be patterned on a polymer surface with a spatial resolution of at least 5 mu m and good reproducibility. MAPS is a multistep procedure: first, the surface of a polymer is modified, in one or more steps, to introduce a reactive group of interest. In a subsequent step, an elastomeric stamp, inked with a biological ligand containing a complementary terminal reactive group, is brought into contact with the activated surface of the polymer. This results in spatially resolved transfer and coupling of the biological ligand to the reactive surface of the polymer. We used MAPS to pattern biotin on carboxylic acid derivatized poly(ethylene terephthalate) (PET), and subsequently with streptavidin, mediated by the high affinity streptavidin-biotin interaction. X-ray photoelectron spectroscopy of biotin-derivatized PET showed that approximately one in five PET repeat units in the top 50-100 Angstrom were functionalized with biotin. Time-of-flight secondary ion mass spectrometry (TOF-SIMS) suggested an increased concentration of PET oligomers in the top 10 Angstrom due to chain scission during modification and clearly identified the derivatization of PET with biotin. TOF-SIMS imaging mapped biotin and streptavidin to the stamped regions. TOF-SIMS also imaged the spatial distribution of residual reagents from the multistep derivatization in MAPS, such as pentafluorophenol, Tween 20 surfactant, as well as poly(dimethylsiloxane) (PDMS), which was transferred from the elastomeric PDMS stamp to the surface during MAPS.