Male zebra finches sing to court females, whereas females do not normally sing. In parallel, the telencephalic brain regions that control song are larger in volume and contain larger cells in males than in females. The vocal control organ (syrinx) is also larger in males. Some evidence suggests that the sexual differentiation of both anatomy and behavior is under the regulation of gonadal hormones during early development, yet recent data conflict with the idea that the sole source of masculinization of the neural song system is the testes. In the present experiment, we treated genetic males with estradiol benzoate on embryonic day 5 and measured the volume of and neuron soma size in robust nucleus of the archistriatum (RA) and the high vocal center (HVC), two telencephalic song control nuclei. We also weighed the syrinx, the muscles of which are the target of the motor pathway containing the two brain regions. The estrogen treatment disrupted testicular morphology, and induced an oviduct in six of seven animals, but it had no effect on any of four measures of masculinization of the neural song system. These results suggest that normal testicular tissue is not required for masculine development of the neural song system.