The vocal control system in many songbird species is a sexually dimorphic neural circuit that mediates learning and production of song. The mechanism by which this system is sexually differentiated has been investigated in only one species, the zebra finch (Taeniopygia guttata). Estradiol may be involved in the sexual differentiation of this system, as female zebra finches treated with estradiol as nestlings develop a male-like song system; however, blocking estradiol action in embryonic and nestling male zebra finches does not demasculinize the song system. Therefore, the role of estradiol in song system development is unclear. The role of estradiol in song system sexual differentiation was assessed in European starlings (Sturnus vulgaris). This species is of potential interest because it is less extreme in the degree of sexual dimorphism of the song system and song behavior than zebra finches. While in the field, starling nestlings were implanted with 500 microg of estradiol at 3 days of age. These birds were brought into the laboratory at Day 11 and hand-reared. In females, estradiol produces significant increases in the volumes of song control regions defined by Nissl stain, as well as by autoradiography for alpha2-adrenergic receptors; however, these estradiol-treated females have song systems that more closely resemble those of control females than control males. Estradiol-treated males exhibit significant hypermasculinization at 210 days of age, but this effect is transient and hypermasculinization is no longer evident at Day 345. The role of estradiol in sexual differentiation of the neural circuit mediating song behavior remains enigmatic.