Amycolata autotrophica converts vitamin D-3(VD3) to 1 alpha,25-dihydroxyvitamin D-3(1 alpha,25(OH)(2)VD3) via 25-hydroxyvitamin D-3(25(OH)VD3) by hydroxylation of VD3 at C-25 and C-1. In this microbial hydroxylation, it was found that cyclodextrin (CD) had the ability to enhance the hydroxylation of VD3. Addition of partially-methylated-beta-cyclodextrin (PMCD) increased the productivity of 25(OH)VD3 about seven-fold compared to that without CD. Combined use of PMCD and gamma-CD increased the production of 1 alpha,25(OH)(2)VD3 in a tank fermenter about sixteen-fold compared to that without CD.