The hydroxylation of testosterone and 5alpha-dihydrotestosterone at C-2 was measured quantitatively in vivo and in vitro in rats by using "metabolically labile" tritium labelled substrates. The enzyme activity was dependent on the sex and age of the animals. There was a good correlation between in vivo and in vitro experiments. Whereas testosterone was hydroxylated only in liver, 5alpha-dihydrotestosterone was hydroxylated in adrenals, kidney, lung and prostate as well. Hydroxylation was not sex-dependent in adrenals and lungs.