We studied the in vivo and in vitro conversion of testosterone to dihydrotestosterone in sebaceous glands microdissected from the costovertebral glands of Syrian hamsters. After the intraperitoneal injection of testosterone- 1,2-3H, dihydrotestosterone was the major radioactive steroid not only in the sebaceous glands but also in the prostate and seminal vesicles. On the other hand, other nontarget organs and the plasma contained very little or no radioactive dihydrotestosterone. Using the newly devised micromethod for 5α-reductase assay with 15 to 25 μg of tissue samples, we found that the sebaceous glands contained sufficient 5α-reductase activity to convert in vivo testosterone to dihydrotestosterone. After castration, the uptake of radioactive dihydrotestosterone after the injection of testosterone- 1,2-3H in the sebaceous glands rapidly decreased, whereas the 5α-reductase activity on a dry tissue weight basis steadily increased. The endogenous level of TPNH, which is essential for 5α-reduction, decreased in the sebaceous gland after castration to nearly 50% of the normal value. These data are consistent with the view that dihydrotestosterone may be the active androgen in the sebaceous gland, as suggested in the prostate and seminal vesicles. Furthermore, the endogenous TPNH level rather than 5α-reductase itself appears to be one of the rate-limiting factors in the control of the dihydrotestosterone level in the sebaceous gland. (Endocrinology90: 73, 1972)