Single-step methods for the generation of patterned surfaces on hydrogels are presented. Poly(vinyl alcohol) films covalently bonded on glass cover slips and commercially available hydrogel-coated polystyrene plates were used as cell-repellent surfaces. Cell-adhesive domains were created by spotting dilute solutions of sodium hypochlorite onto the surfaces. Alternatively, domains supporting cell attachment were created by exposure to UV light from a xenon excimer lamp, employing a contact mask. Rat skeletal myoblast cells, HEK 293 human embryonic kidney cells and Caco-2 colon carcinoma cells adhered and spread exclusively on modified areas. The surfaces are durable for weeks under cell culture conditions and re-usable after removal of the cells by trypsin treatment. Arrays of adhesive spots seeded with cells at a low density permitted dynamic monitoring of cell proliferation. Selected colonies can be harvested from the surfaces by means of local trypsination. Thus, these techniques may provide useful tools for the isolation of clonal cell populations. Additionally, we demonstrate the possibility of surface-mediated gene delivery from the micro patterns. We show that DNA, complexed with a lipid reagent, can be adsorbed on modified poly(vinyl alcohol) coatings, resulting in spatially controlled adhesion and reverse transfection of HEK 293 cells.