Hydrogel microstructures with micrometer-scale topography and controllable functionality have great potential for numerous nanobiotechnology applications including, for example, three-dimensional structures that exhibit controlled interactions with proteins and cells. Taking advantage of the strong affinity of histidine (His) residues for metal-ion-nitrilotriacetic acid (NTA) complexes, we have chemically modified hydrogels to enable protein immobilization with retention of activity by incorporating 2-methacrylamidobutyl nitrilotriacetic acid, an NTA-containing monomer that can be copolymerized with a series of monomers to form NTA-containing hydrogels. By varying the NTA-monomer composition in the hydrogels, it is possible to control the amount of protein bound to the hydrogel surface. He retention of biological activity was demonstrated by microtubule gliding assays. Normally, hydrogels are resistant to protein binding, but we have selected these materials because of their porous nature. Bringing together hydrogel functionalization and soft-lithography patterning techniques, it was possible to create a hybrid hydrogel superstructure that possesses binding specificity to His-tagged protein in selected sites. This type of surface and microstructure is not oaly advantageous for motor protein integration, but it can also be generally applied to the formation of His-tagged molecules for sensors and biochip applications. © 2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.