Although there is growing evidence that estradiol modulates female perception of male sexual signals, relatively little research has focused on female auditory processing. We used in vivo 18F-fluorodeoxyglucose (18F-FDG) positron emission tomography (PET) imaging to examine the neuronal effects of estradiol and conspecific song in female house sparrows (Passer domesticus). We assessed brain glucose metabolism, a measure of neuronal activity, in females with empty implants, estradiol implants, and empty implants ~1 month after estradiol implant removal. Females were exposed to conspecific or heterospecific songs immediately prior to imaging. The activity of brain regions involved in auditory perception did not differ between females with empty implants exposed to conspecific vs. heterospecific song, but neuronal activity was significantly reduced in females with estradiol implants exposed to heterospecific song. Furthermore, our within-individual design revealed that changes in brain activity due to high estradiol were actually greater several weeks after peak hormone exposure. Overall, this study demonstrates that PET imaging is a powerful tool for assessing large-scale changes in brain activity in living songbirds, and suggests that after breeding is done, specific environmental and physiological cues are necessary for estradiol-stimulated females to lose the selectivity they display in neural response to conspecific song.