A microarray chip containing human P450 isoforms was constructed for the parallel assay of their metabolic activities. The chip had microwells that contained vertically integrated P450 and oxygen sensing layers. The oxygen sensing film was made of an organically modified silica film (ORMOSIL) doped with tris(4,7-diphenyl-1,10-phenanthroline) ruthenium dichloride (Ru(dpp)(3)Cl(2)). Human P450s (23 types) expressed in E. coli and purified as membrane fractions were immobilized in agarose matrixes on the oxygen sensing layer. The activities of P450s were determined by evaluating the fluorescence intensity enhancement of the oxygen sensor due to the oxygen consumption by the metabolic reaction. By normalizing the responses with the amounts of oxygen sensor and P450 enzymes in microwells, we could obtain fluorescence enhancement patterns that were characteristic to the combination of P450 isoforms and substrate material. The patterns obtained from two psoralen derivatives resembled each other, whereas a structurally different substrate (capsaicin) resulted in a distinct pattern. These results suggest the potential of the microarray to analyze the activities of diverse P450 isoforms in a high-throughput fashion. Furthermore, mechanism-based inactivation (MBI) of P450 could be detected by successively incubating a chip with different substrate solutions and measuring the residual activities.