A combination of nanoimprint lithography and microcontact printing was used to create cell substrates with well-defined nanotopographic patterns of grooves overlaid with independently controlled micropatterned chemical domains. Qualitative analysis of osteoblast-like cells cultured on the substrates showed alignment of cells and cell features to the nanotopographic grooves when surface chemistry was either uniform or a pattern of dots. When surface chemistry on the substrate was a pattern of lanes, cells aligned to the lanes. On all substrates, small cellular extensions, or filopodia, displayed no particular alignment to either nanotopographic grooves or chemical patterns. Large cell extensions were observed only parallel to either nanotopographic grooves or chemical lanes. The techniques used provide an easily scaleable approach to creating cell substrates that will aid in studying the relative impact and interplay of chemical patterns and mechanical topography on cellular responses. (c) 2005 American Vacuum Society.