1α,25-Dihydroxyvitamin D 3 [1α,25(OH) 2 D 3 ] is mainly metabolized via the C-24 oxidation pathway and undergoes several side chain modifications which include C-24 hydroxylation, C-24 ketonization, C-23 hydroxylation and side chain cleavage between C-23 and C-24 to form the final product, calcitroic acid. In a recent study we reported that 1α,25-dihydroxyvitamin D 2 [1α,25(OH) 2 D 2 ] like 1α,25(OH) 2 D 3 , is also converted into the same final product, calcitroic acid. This finding indicated that 1α,25(OH) 2 D 2 also undergoes side chain cleavage between C-23 and C-24. As the side chain of 1α,25(OH) 2 D 2 when compared to the side chain of 1α,25(OH) 2 D 3 , has a double bond between C-22 and C-23 and an extra methyl group at C-24 position, it opens the possibility for both (a) double bond reduction and (b) demethylation to occur during the metabolism of 1α,25(OH) 2 D 2 . We undertook the present study to establish firmly the possibility of double bond reduction in the metabolism of vitamin D 2 related compounds. We compared the metabolism of 1α,25-dihydroxy-22-ene-vitamin D 3 [1α,25(OH) 2 -22-ene-D 3 ], a synthetic vitamin D analog whose side chain differs from that of 1α,25(OH) 2 D 3 only through a single modification namely the presence of a double bond between C-22 and C-23. Metabolism studies were performed in the chronic myeloid leukemic cell line (RWLeu-4) and in the isolated perfused rat kidney. Our results indicate that both 1α,25(OH) 2 -22-ene-D 3 and 1α,25(OH) 2 D 3 are converted into common metabolites namely, 1α,24(R),25-trihydroxyvitamin D 3 [1α,24(R),25(OH) 3 D 3 ], 1α,25-dihydroxy-24-oxovitamin D 3 [1α,25(OH) 2 -24-oxo-D 3 ], 1α,23(S),25-trihydroxy-24-oxovitamin D 3 and 1α,23-dihydroxy-24,25,26,27-tetranorvitamin D 3 . This finding indicates that the double bond in the side chain of 1α,25(OH) 2 -22-ene-D 3 is reduced during its metabolism. Along with the aforementioned metabolites, 1α,25(OH) 2 -22-ene-D 3 is also converted into two additional metabolites namely, 1α,24,25(OH) 3 -22-ene-D 3 and 1α,25(OH) 2 -24-oxo-22-ene-D 3 . Furthermore, we did not observe direct conversion of 1α,25(OH) 2 -22-ene-D 3 into 1α,25(OH) 2 D 3 . These findings indicate that 1α,25(OH) 2 -22-ene-D 3 is first converted into 1α,24,25(OH) 3 -22-ene-D 3 and 1α,25(OH) 2 -24-oxo-22-ene-D 3 . Then the double bonds in the side chains of 1α,24,25(OH) 3 -22-ene-D 3 and 1α,25(OH) 2 -24-oxo-22-ene-D 3 undergo reduction to form 1α,24(R),25(OH) 3 D 3 and 1α,25(OH) 2 -24-oxo-D 3 , respectively. Thus, our study indicates that the double bond in 1α,25(OH) 2 -22-ene-D 3 is reduced during its metabolism. Furthermore, it appears that the double bond reduction occurs only during the second or the third step of 1α,25(OH) 2 -22-ene-D 3 metabolism indicating that prior C-24 hydroxylation of 1α,25(OH) 2 -22-ene-D 3 is required for the double bond reduction to occur.