This report describes a method for patterning ligands onto mixed SAMs of alkanethiolates on gold by microcontact printing (mu CP). The mixed SAMs were made from thiols presenting terminal tri(ethylene glycol) groups (HS(CH2)(11)(OCH2CH2)(3)OH, 1) and terminal hexa(ethylene glycol)-CH2CO2H groups (HS(CH2)(11)(OCH2CH2)(6)OCH2CO2H, 2). Ligands were printed using a two-step procedure. The carboxylic acid groups of 2 were first converted to reactive pentafluorophenyl esters. A freshly oxidized PDMS stamp, inked with a ligand derivatized with a primary amine, was then brought into contact with the activated SAM; in the areas of contact, the amine reacted with the activated. ester and formed an amide. Two ligands, biotin and benzenesulfonamide, were printed onto these SAMs. The formation of patterned SAMs presenting biotin ligands was detected by fluorescence microscopy of substrates that were incubated with a solution of fluorescently labeled antibiotin antibody. The formation of patterned biotin was also detected using a sandwich experiment; in this experiment, the SAM was incubated sequentially in solutions of streptavidin, protein G-biotin conjugate, and fluorescently labeled goat antirabbit IgG. The smallest features resolved in images obtained by these methods were squares with a 5 mu um side. Using surface plasmon resonance (SPR) to detect binding of antibiotin antibody to SAMs presenting biotin groups, the yield of coupling by mu CP was estimated to be similar to 90% of that obtained by immersion. Printing of the benzenesulfonamide ligand was detected by binding of carbonic anhydrase (CA) to the sulfonamide-derivatized SAMs; the yield of coupling, as estimated by SPR, was similar to 75% of that obtained by immersion. For both ligands, oxidation of the PDMS stamp before inking was found to be critical for good coupling yields.