Our previous studies revealed that the double variants of CYP105A1- R73A/R84A and R73V/R84A-show high levels of activity with respect to conversion of vitamin D-3 to its biologically active form, 1 alpha,25-dihydroxyvitamin D-3 (1 alpha,25(OH)(2)D-3). In this study, we found that both the double variants were also capable of converting vitamin D-2 to its active form, that is, 1 alpha,25-dihydroxyvitamin D-2 (1 alpha,25(OH)(2)D-2), via 25(OH)D-2, whereas its 1 alpha-hydroxylation activity toward 25(OH)D-2 was much lower than that toward 25(OH)D-3. Comparison of the wild type and the double variants revealed that the amino acid substitutions remarkably enhanced both 25- and 26-hydroxylation activity toward vitamin D-2. After 25-hydroxylation of vitamin D-2, further hydroxylation at C26 may occur frequently without the release of 25(OH)D-2 from the substrate-binding pocket. Thus, the double variants of CYP105A1 are quite useful to produce 25,26(OH)(2)D-2 that is one of the metabolites of vitamin D2 detected in human serum. (C) 2016 Elsevier Inc. All rights reserved.