Pseudonocardia autotrophica converted Vitamin D 3 to 25-hydroxyvitamin D 3 and 1α,25-dihydroxyvitamin D 3 . The hydroxylation of Vitamin D 3 with P. autotrophica was enhanced by the addition of cyclodextrin. In this microbial hydroxylation, a new Vitamin D 3 metabolite was observed in the reaction mixture of P. autotrophica and Vitamin D 3 , and was isolated in a pure form by several steps of chromatography. The structure of the new metabolite was determined to be 2α,25-dihydroxyvitamin D 3 by UV, NMR and mass spectroscopic analyses. Biological evaluation of the new metabolite was conducted by means of several experiments.