Human 25‐hydroxyvitamin D₃ (25(OH)D₃) 24‐hydroxylase (CYP24) cDNA was expressed in Escherichia coli, and its enzymatic and spectral properties were revealed. The reconstituted system containing the membrane fraction prepared from recombinant E. coli cells, adrenodoxin and adrenodoxin reductase was examined for the metabolism of 25(OH)D₃, 1α,25(OH)₂D₃ and their related compounds. Human CYP24 demonstrated a remarkable metabolism consisting of both C‐23 and C‐24 hydroxylation pathways towards both 25(OH)D₃ and 1α,25(OH)₂D₃, whereas rat CYP24 showed almost no C‐23 hydroxylation pathway [Sakaki, T. Sawada, N. Nonaka, Y. Ohyama, Y. & Inouye, K. (1999) Eur. J. Biochem. 262, 43–48]. HPLC analysis and mass spectrometric analysis revealed that human CYP24 catalyzed all the steps of the C‐23 hydroxylation pathway from 25(OH)D₃ via 23S,25(OH)₂D₃, 23S,25,26(OH)₃D₃ and 25(OH)D₃‐26,23‐lactol to 25(OH)D₃‐26,23‐lactone in addition to the C‐24 hydroxylation pathway from 25(OH)D₃ via 24R,25(OH)₂D₃, 24‐oxo‐25(OH)D₃, 24‐oxo‐23S,25(OH)₂D₃ to 24,25,26,27‐tetranor‐23(OH)D₃. On 1α,25(OH)₂D₃ metabolism, similar results were observed. These results strongly suggest that the single enzyme human CYP24 is greatly responsible for the metabolism of both 25(OH)D₃ and 1α,25(OH)₂D₃. We also succeeded in the coexpression of CYP24, adrenodoxin and NADPH‐adrenodoxin reductase in E. coli. Addition of 25(OH)D₃ to the recombinant E. coli cell culture yielded most of the metabolites in both the C‐23 and C‐24 hydroxylation pathways. Thus, the E. coli expression system for human CYP24 appears quite useful in predicting the metabolism of vitamin D analogs used as drugs.