In our previous study, we indicated for the first time that C-28 hydroxylation plays a significant role in the metabolism of 1α,25-dihydroxyvitamin D 2 [1α,25(OH) 2 D 2 ] by identifying 1α,24(S),25,28-tetrahydroxyvitamin D 2 [1α,24(S),25,28(OH) 4 D 2 ] as a major renal metabolite of 1α,25(OH) 2 D 2 [G. S. Reddy and K-Y. Tserng Biochemistry 25, 5328-5336, 1986]. The present study was performed to establish the physiological significance of C-28 hydroxylation in the metabolism of 1α,25(OH) 2 D 2 . We perfused rat kidneys in vitro with 1α,25(OH) 2 [26,27- 3 H]D 2 (5 x 10 - 1 0 M) and demonstrated that 1α,24(R),25-trihydroxyvitamin D 2 [1α,24(R),25(OH) 3 D 2 ] and 1α,24(S),25,28(OH) 4 D 2 are the only two major physiological metabolites of 1α,25(OH) 2 D 2 . In the same perfusion experiments, we also noted that there is no conversion of 1α,25(OH) 2 D 2 into 1α,25,28-trihydroxyvitamin D 2 [1α,25,28(OH) 3 D 2 ]. Moreover, 1α,24(S),25,28(OH) 4 D 2 is not formed in the perfused rat kidney when synthetic 1α,25,28(OH) 3 D 2 is used as the starting substrate. This finding indicates that C-28 hydroxylation of 1α,25(OH) 2 D 2 occurs only after 1α,25(OH) 2 D 2 is hydroxylated at C-24 position. At present the enzyme responsible for the C-28 hydroxylation of 1α,24(R),25(OH) 3 D 2 in rat kidney is not known. Recently, it was found that 1α,25(OH) 2 D 3 -24-hydroxylase (CYP24) can hydroxylate carbons 23, 24, and 26 of various vitamin D 3 compounds. Thus, it may be speculated that CYP24 may also be responsible for the C-28 hydroxylation of 1α,24(R),25(OH) 3 D 2 to form 1α,24(S),25,28(OH) 4 D 2 . The biological activity of 1α,24(S),25,28(OH) 4 D 2 , determined by its ability to induce intestinal calcium transport and bone calcium resorption in the rat, was found to be almost negligible. Also, 1α,24(S),25,28(OH) 4 D 2 exhibited very low binding affinity toward bovine thymus vitamin D receptor. These studies firmly establish that C-28 hydroxylation is an important enzymatic reaction involved in the inactivation of 1α,25(OH) 2 D 2 in kidney under physiological conditions.