Thimet oligopeptidase (EC 3.4.24.15; also called EP24.15 and TOP; referred to here as TOP) is a neuropeptidase involved in the regulation of several physiological functions including reproduction. Among its substrates is gonadotrophin-releasing hormone (GnRH), an important hypothalamic hormone that regulates the synthesis and release of oestradiol and facilitates female sexual behaviour. Using immunohistochemistry, we found that TOP is expressed in the nucleus of cells throughout the female mouse brain, and in high levels in steroid-sensitive regions of the hypothalamus, which is consistent with previous findings in male rats. Furthermore, dual-label immunofluorescence revealed that TOP and oestrogen receptor alpha (ERalpha) coexpress in several reproductively-relevant brain regions, including the medial preoptic area (mPOA), arcuate nucleus (ARC), ventrolateral portion of the ventromedial hypothalamic nucleus (VMNvl) and the midbrain central grey (MCG). Previous studies in rats have shown that oestradiol decreases hypothalamic TOP levels or activity, possibly potentiating the effects of GnRH. In the present study, analysis by immunohistochemistry revealed that oestradiol decreased TOP immunoreactivity in the VMNvl, whereas no differences were detected in the mPOA, ARC or median eminence. Overall, the present findings indicate that TOP is coexpressed with ERalpha, and oestradiol regulates TOP expression in a brain region-specific manner in female mice, providing neuroanatomical evidence that TOP may function in reproductive physiology and/or behaviour.