In the brain of medaka (Oryzias latipes), a teleost fish, we recently found that the supracommissural/posterior nuclei of the ventral telencephalic area (Vs/Vp) and the magnocellular/gigantocellular portions of the magnocellular preoptic nucleus (PMm/PMg) express estrogen receptor (ER) and androgen receptor (AR) specifically in females. This finding led us to postulate that sex steroid hormones might induce gene expression unique to females in these nuclei. In the present study, we searched for genes differentially expressed between the sexes in the medaka brain and identified the gene encoding neuropeptide B (npb) as being female-specifically expressed in Vs/Vp and PMm/PMg. As expected, the neurons expressing npb female-specifically constituted a significant proportion of the ER/AR-expressing neurons in these nuclei. Subsequent analyses provided evidence that the female-specific expression of npb in Vs/Vp and PMm/PMg results from the reversible and transient action of estrogens secreted from the ovary and that this estrogenic action is most likely mediated by the direct transcriptional activation of npb through an estrogen-responsive element in its proximal promoter region. Vs/Vp and PMm/PMg are generally recognized in teleost fish as the sites where neurons expressing 2 other neuropeptides, isotocin and vasotocin, are present, but the female-specific npb/ER/AR-expressing neurons were distinct from, although adjacent to, isotocin and vasotocin neurons. Taken together, these data demonstrate that npb is female-specifically expressed in novel, as-yet undefined populations of Vs/Vp and PMm/PMg neurons, resulting from the direct stimulatory action of ovarian estrogens via female-specific ER in these neurons.