Genetic variations in cytochrome P450 2C9 (CYP2C9) contribute to interindividual variability in the metabolism of clinically used drugs such as warfarin and tolbutamide. We functionally characterized 32 types of allelic variant CYP2C9 proteins. Recombinant CYP2C9 proteins generated using a heterologous expression system are useful for comparing functional changes in CYP2C9 variant proteins expressed from low-frequency alleles. Wild-type CYP2C9 and its 31 variants were found to be transiently expressed in COS-7 cells, and the enzymatic activity of the CYP2C9 variants was characterized using S-warfarin as a representative substrate. Among the 32 types of CYP2C9 allelic variants tested, CYP2C9.18, CYP2C9.21, CYP2C9.24, CYP2C9.26, CYP2C9.33 and CYP2C9.35 exhibited no enzyme activity, and 12 types showed significantly decreased enzyme activity. In vitro analysis of CYP2C9 variant proteins should be useful for predicting CYP2C9 phenotypes and for application to personalized drug therapy.