Androgens and estrogens influence the maturation and function of numerous tissues in both male and female birds, especially the brains of the oscine songbirds. Although there exist a very large number of studies that have investigated circulating sex steroids in many species of wild and captive-held songbirds, there remain a significant number of questions about the sites of synthesis of the active steroids that act on the songbird brain. Estrogens are derived from androgen. Thus, the synthesis of androgen itself is critical for both androgen- and estrogen-dependent actions in both male and female songbirds. Therefore, we have undertaken studies of the enzyme 17alpha-hydroxylase/C17,20-lyase (Cyp17), the enzyme responsible for the synthesis of androgens from their progestin or pregnane precursors via their 17alpha-hydroxy intermediates. Here we have characterized optimal conditions for measuring Cyp17 in gonads of adult zebra finches via the conversion of tritiated [3H]progesterone into 17alpha-hydroxy P (17alpha-hydroxylase activity) and androstenedione and testosterone (C17,20-lyase) activity. Cyp17 activity is abundant in testis, with lesser amounts in ovary. Low levels of Cyp17 activity were also detected in male adrenals, but not in any other tissue, including brain. Testicular Cyp17 activity is readily inhibited in vitro by ketoconazole, a specific Cyp17 inhibitor. Ketoconazole works less well in vivo. In males castrated and/or treated with fadrozole, an inhibitor of aromatase, we detected no extragonadal sites of Cyp17 activity, although fadrozole appeared to increase circulating androgens in both castrated and intact males. Thus, we still do not know the site of androgen synthesis in these males. Further studies of Cyp17 will be useful in understanding more about the mechanisms of androgen delivery to neural circuits in adult and developing songbirds.