The compound 26,26,26,27,27,27-F6-1α,25(OH)2D3 is a hexafluorinated analog of the active form of Vitamin D3. The enhanced biological activity of F6-1α,25(OH)2D3 is considered to be related to a decreased metabolic inactivation of the compound in target tissues such as the kidneys, small intestine, and bones. Our previous study demonstrated that CYP24 is responsible for the metabolism of F6-1α,25(OH)2D3 in the target tissues. In this study, we compared the human and rat CYP24-dependent metabolism of F6-1α,25(OH)2D3 by using the Escherichia coli expression system. In the recombinant E. coli cells expressing human CYP24, bovine adrenodoxin and NADPH-adrenodoxin reductase, F6-1α,25(OH)2D3 was successively converted to F6-1α,23S,25(OH)3D3, F6-23-oxo-1α,25(OH)2D3, and the putative ether compound with the same molecular mass as F6-1α,25(OH)2D3. The putative ether was not observed in the recombinant E. coli cells expressing rat CYP24. These results indicate species-based difference between human and rat CYP24 in the metabolism of F6-1α,25(OH)2D3. In addition, the metabolite with a cleavage at the C24&z.sbnd;C25 bond of F6-1α,25(OH)2D3 was detected as a minor metabolite in both human and rat CYP24. Although F6-1α,23S,25(OH)3D3 and F6-23-oxo-1α,25(OH)2D3 had a high affinity for Vitamin D receptor, the side-chain cleaved metabolite and the putative ether showed extremely low affinity for Vitamin D receptor. These findings indicate that human CYP24 has a dual pathway for metabolic inactivation of F6-1α,25(OH)2D3 while rat CYP24 has only one pathway. Judging from the fact that metabolism of F6-1α,25(OH)2D3 in rat CYP24-harboring E. coli cells is quite similar to that in the target tissues of rat, the metabolism seen in human CYP24-harboring E. coli cells appear to exhibit the same metabolism as in human target tissues. Thus, this recombinant system harboring human CYP24 appears quite useful for predicting the metabolism and efficacy of Vitamin D analogs in human target tissues before clinical trials. [Copyright &y& Elsevier]