The metabolism of 1 alpha,25-dihydroxyvitamin D-2 (1 alpha,25(OH)(2)D-2) by human CYP24A1 was examined using the recombinant enzyme expressed in Escherichia coli cells. HPLC analysis revealed that human CYP24A1 produces at least 10 metabolites, while rat CYP24A1 produces only three metabolites, indicating a remarkable species-based difference in the CYP24A1-dependent metabolism of 1 alpha,25(OH)(2)D-2 between humans and rats. LC-MS analysis and periodate treatment of the metabolites strongly suggest that human CYP24A1 converts 1 alpha,25(OH)(2)D-2 to 1 alpha,24,25,26(OH)(4)D-2, 1 alpha,24,25,28(OH)(4)D-2, and 24-oxo-25,26,27-trinor-1 alpha(OH)D-2 Via 1 alpha,24,25(OH)(3)D-2. These results indicate that human CYP24A1 catalyzes the C-24-C-25 bond cleavage of 1 alpha,24,25(OH)(2)D-2, which is quite effective in the inactivation of the active form of vitamin D-2. The combination of hydroxylation at multiple sites and C-C bond cleavage could form a large number of metabolites. Our findings appear to be useful to predict the metabolism of vitamin D-2 and its analogs in the human body. (C) 2009 Elsevier Inc. All rights reserved.