The Kiss1 gene codes for kisspeptins, which have been implicated in the neuroendocrine regulation of reproduction. In the brain, Kiss1 mRNA-expressing neurons are located in the arcuate (ARC) and anteroventral periventricular (AVPV) nuclei. Kiss1 neurons in the AVPV appear to play a role in generating the preovulatory GnRH/LH surge, which occurs only in females and is organized perinatally by gonadal steroids. Because Kiss1 is involved in the sexually dimorphic GnRH/LH surge, we hypothesized that Kiss1 expression is sexually differentiated, with females having more Kiss1 neurons than either males or neonatally androgenized females. To test this, male and female rats were neonatally treated with androgen or vehicle; then, as adults, they were left intact or gonadectomized and implanted with capsules containing sex steroids or nothing. Kiss1 mRNA levels in the AVPV and ARC were determined by in situ hybridization. Normal females expressed significantly more Kiss1 mRNA in the AVPV than normal males, even under identical adult hormonal conditions. This Kiss1 sex difference was organized perinatally, as demonstrated by the observation that neonatally androgenized females displayed a male-like pattern of adulthood Kiss1 expression in the AVPV. In contrast, there was neither a sex difference nor an influence of neonatal treatment on Kiss1 expression in the ARC. Using double-labeling techniques, we determined that the sexually differentiated Kiss1 neurons in the AVPV are distinct from the sexually differentiated population of tyrosine hydroxylase (dopaminergic) neurons in this region. Our findings suggest that sex differences in kisspeptin signaling from the AVPV subserve the cellular mechanisms controlling the sexually differentiated GnRH/LH surge.