The 20-epi form of 1alpha,25-dihydroxyvitamin D-3 (1alpha,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 1alpha,25-dihydroxyvitamin D3 while its calcemic activity is only slightly elevated. In this study, we compared the human and rat CYP24-dependent metabolism of 1alpha,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 1alpha,25(OH)(2)-20-epi-D-3 to 25,26,27-trinor-1alpha(OH)-24(COOH)-20-epi-D-3 through 1alpha,24,25(OH)(3)-20-epi-D-3 and 1alpha,25(OH)(2)-24-oxo-20-epi-D-3. The binding affinity of trinor-1alpha(OH)-24(COOH)-20-epi-D-3 for vitamin D receptor (VDR) was less than 1/4000 of that of 1alpha,25(OH)(2)-20-epi-D-3. These results suggest that rat CYP24 can almost completely inactivate 1alpha,25(OH)(2)-20-epi-D-3. On the other hand, human CYP24 mainly converted 1alpha,25(OH)(2)-20-epi-D-3 to its putative demethylated compound with a hydroxyl group, via 1alpha,24,25(OH)(3)-20-epi-D-3, 1alpha,25(OH)(2)-24-oxo-20-epi-D-3, and 1alpha,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 lot,25(OH)2-20-epi-D-3. (C) 2003 Elsevier Inc. All rights reserved.