CYP2R1 is known to be a physiologically important vitamin D 25-hydroxylase. We have successfully expressed human CYP2R1 in Saccharomyces cerevisiae to reveal its enzymatic properties. In this study, we examined production of 25-hydroxylated vitamin D using whole recombinant yeast cells that expressed CYP2R1. When vitamin D-3 or vitamin D-2 was added to the cell suspension of CYP2R1-expressing yeast cells in a buffer containing glucose and B-cyclodextrin, the vitamins were converted into their 25-hydroxylated products. Next, we irradiated the cell suspension with UVB and incubated at 37 degrees C. Surprisingly, the 25-hydroxy vitamin D-2 was produced without additional vitamin Dy. Endogenous ergosterol was likely converted into vitamin D-2 by UV irradiation and thermal isomerization, and then the resulting vitamin D-2 was converted to 25-hydroxyvitamin D-2 by CYP2R1. This novel method for producing 25-hydroxyvitamin D-2 without a substrate could be useful for practical purposes. (C) 2013 Elsevier Inc. All rights reserved.