In order to evaluate the potential adverse effects due to genetic polymorphism and/or inter-individual variation, it is necessary to calculate the cytochrome P450 (CYP) contribution to the metabolism of new drugs. In the current study, the in vitro intrinsic clearance (CL(int)) values of marker substrates and drugs were determined by measuring metabolite formation and substrate depletion, respectively. Recombinant CYP microsomes expressing CYP2C9, CYP2C19 and CYP3A4 with co-expressed cytochrome b(5) were used, but those expressing CYP1A2 and CYP2D6 did not have co-expressed cytochrome b(5). The following prediction methods were compared to determine the CL(int) value using data from recombinant CYP enzymes: (1) relative CYP enzyme content in human liver microsomes; (2) relative activity factor (RAF) estimated from the V(max) value; and (3) RAF estimated from the CL(int) value. Estimating RAF from CL(int) proved the most accurate prediction method among the three tested, and differences in the CYP3A4 marker reactions did not affect its accuracy. The substrate depletion method will be useful in the early drug-discovery stage when the main metabolite and/or metabolic pathway has not been identified. In addition, recombinant CYP microsomes co-expressed with cytochrome b(5) might be suitable for the prediction of the CL(int) value.