Human vitamin D-3 25-hydroxylase (CYP27A1) cDNA was expressed in Escherichia coli, and its enzymatic properties were revealed. The reconstituted system containing the membrane fraction prepared from the recombinant E. coli cells was examined for the metabolism of vitamin Ds. Surprisingly, at least eight forms of metabolites including the major product 25(OH)D-3 were observed. HPLC analysis and mass spectrometric analysis suggested that those metabolites were 25(OH)D-3, 26(OW)D-3, 27(OH)D-3, 24R,25(OH)(2)D-3, 1 alpha,25(OH)(2)D-3, 25,26(OH)(2)D-3 (25,27(OH)(2)D-3), 27-oxo-D-3 and a dehydrogenated form of vitamin D-3. These results suggest that human CYP27A1 catalyzes multiple reactions and multiple-step metabolism toward vitamin D-3. The K-m and V-max values for vitamin D-3 25-hydroxylation and 25(OH)D-3 1 alpha-hydroxylation were estimated to be 3.2 mu M and 0.27 (mol/min/mol P450), and 3.5 mu M and 0.021 (mol/min/mol P450), respectively. These kinetic studies have made it possible to evaluate a physiological meaning of each reaction catalyzed by CYP27A1.