We report on a methodology for the covalent immobilization of human CYP2E1 and glucose-6-phosphate dehydrogenase (G6PD) in the microchannels of porous alumina membranes (60 mu m thickness) for the purpose of xenobiotic metabolic's studies. The natural packing of porous alumina in aligned microchannels allow high loading of enzymes in the form of thin film as compared to flat support of the same projected area. A system was developed by stacking two independently modified membranes of silane-glutaraldehyde and silane-maleimide containing immobilized G6PD and recombinant human cytochrome CYP2E1 respectively. A more complex two-step enzyme reaction was designed by placing the membranes in a fluidic device at fast flow rates which affords short residence times even in second(s) are sufficient to get substrate transformation. Product formed by G6PD modified membrane became the cofactor for CYP2E1, patterned downstream, where fluorogenic substrate turnover was recorded. (c) 2008 Elsevier B.V. All rights reserved.