When estradiol-17 beta 17-glucuronide was incubated with male rat liver microsomal preparations with a NADPH-generating system, 2-hydroxyestradiol-17 beta 17-glucuronide was obtained. This 2-hydroxylation was shown to occur without cleavage of the conjugate group. The result clearly indicates that estradiol-17 beta 17-glucuronide could act as substrate for rat liver microsomal 2-hydroxylase.