The 20-epi form of 1α,25-dihydroxyvitamin D 3 (1α,25(OH) 2-20-epi-D 3) is expected as drugs for leukemia, other cancers or psoriasis, because it shows several-hundred fold enhanced ability to induce cell differentiation and growth inhibition than 1α,25-dihydroxyvitamin D 3 while its calcemic activity is only slightly elevated. In this study, we compared the human and rat CYP24-dependent metabolism of 1α,25(OH) 2-20-epi-D 3 by using the Escherichia coli expression system. The HPLC and LC-MS analyses of the metabolites revealed that rat CYP24 converted 1α,25(OH) 2-20-epi-D 3 to 25,26,27-trinor-1α(OH)-24(COOH)-20-epi-D 3 through 1α,24,25(OH) 3-20-epi-D 3 and 1α,25(OH) 2-24-oxo-20-epi-D 3. The binding affinity of trinor-1α(OH)-24(COOH)-20-epi-D 3 for vitamin D receptor (VDR) was less than 1/4000 of that of 1α,25(OH) 2-20-epi-D 3. These results suggest that rat CYP24 can almost completely inactivate 1α,25(OH) 2-20-epi-D 3. On the other hand, human CYP24 mainly converted 1α,25(OH) 2-20-epi-D 3 to its putative demethylated compound with a hydroxyl group, via 1α,24,25(OH) 3-20-epi-D 3, 1α,25(OH) 2-24-oxo-20-epi-D 3, and 1α,23,25(OH) 3-24-oxo-20-epi-D 3. All of these metabolites showed considerable affinity for vitamin D receptor. These results clearly demonstrate the species-based difference between human and rat on the CYP24-dependent metabolism of 1α,25(OH) 2-20-epi-D 3.