Unique reproductive phenomena, such as delayed fertilization in females and asynchrony between spermatogenesis and mating behavior in males, are known in hibernating bats. The present study was undertaken to examine sex differences and seasonal changes in the gonadotropin-releasing hormone (GnRH)-immunoreactive (ir) neuronal system of Japanese bats, Rhinolophus ferrumequinum. GnRH-ir neurons were preferentially distributed in the medial preoptic area (POA) and medial basal hypothalamus (MBH). The number and immunoreactivity of GnRH-ir neurons decreased in summer (lactation period in females and spermatogenic period in males), whereas both increased dramatically in winter (hibernation period). The number and immunoreactivity of GnRH-ir neurons varied more in the MBH than in the POA throughout the annual reproductive cycle of both sexes. The changes in GnRH neurons in the MBH closely paralleled those of GnRH-ir fibers in the median eminence (ME), which is the release site for GnRH. In contrast, there was no sex difference in the number and immunoreactivity of GnRH-ir neuronal perikarya in either region except for the number of GnRH-ir neurons in the POA in spring and summer. These findings suggest that GnRH neurons in the MBH supply the major GnRH innervation to the ME and play a central role in seasonal regulation of gonadotropin secretion in this bat.