The avian song control system is an excellent model in which to study the effects of gonadal steroid hormones on neural and behavioral plasticity. Several of the brain regions that control song behavior concentrate androgens and/or estrogens. Investigations of the distribution and regulation of androgen receptors have been limited by the lack of a reliable immunocytochemical method to detect androgen receptors in the songbird brain. We describe a protocol by which the PG-21 polygonal antibody to the rat androgen receptor can be used to label androgen receptor-containing cells in the songbird brain. By treating songbirds of several species with testosterone 90 min before sacrifice and by using relatively low concentrations (0.5 0.75 microg/ml) of PG-21 antibody to reduce nonspecific background staining, we were able to obtain strong specific labeling of cell nuclei in androgen-sensitive brain regions. This technique will facilitate the study of the role of androgens in mediating neural plasticity in the avian brain. Testosterone pretreatment may also facilitate the use of this antibody to label androgen receptors in tissues from a wide array of nonmammalian species.