Nanoporous aluminum oxide membranes were prepared by anodic oxidation of aluminum for application as novel cell culture substrates. Self-supporting as well as mechanically stabilized nanoporous membranes were produced from aluminum plates and micro-imprinted aluminum foils, respectively. Membranes of two different pore sizes (70 and 260 nm) were selected to investigate cellular interactions with such nanoporous substrates using cells of hepatoma cell line HepG2. The membranes express excellent cell-growth conditions. As shown by scanning electron microscopy investigations, the cells could easily adhere to the membranes and proliferate during a 4 day cell culture period. The cells exhibit normal morphology and were able to penetrate into pores with a diameter of 260 nm by small extensions (filopodia). On mechanically stabilized aluminum oxide membranes it was observed that the cells even adhere to the walls of the small cavities. It was demonstrated experimentally that the nanoporous aluminum oxide membranes are well suited as substrates in cell culture model systems for metabolic, pharmacological/toxicological research, tissue engineering and studies on pathogens as well as bioartificial liver systems.