In rodents, kisspeptin-expressing neurons are localized in 2 hypothalamic brain nuclei (anteroventral periventricular nucleus/periventricular nucleus continuum [AVPv/PeN] and arcuate nucleus [ARC]) and modulated by sex steroids. By using wild-type (WT) and aromatase knockout (ArKO) mice (which cannot convert testosterone into estradiol) and immunohistochemistry, we observed that WT females showed a continuous increase in kisspeptin peptide expression in the ARC across postnatal ages (postnatal day 5 [P5] to P25), whereas WT males did not show any expression before P25. Kisspeptin peptide expression was also present in ArKO females but did not increase over this early postnatal period, suggesting that kisspeptin peptide expression in the ARC is organized by estradiol-dependent and -independent mechanisms. We also compared kisspeptin peptide expression between groups of adult male and female mice that were left gonadally intact or gonadectomized and treated or not with estradiol (E(2)) or DHT. In the ARC, kisspeptin peptide expression decreased after gonadectomy but was completely rescued by either E(2) or DHT treatment in each sex/genotype. However, kisspeptin peptide expression was lower in ArKO compared with WT subjects. In the AVPv/PeN, ArKO females showed a male-typical kisspeptin peptide expression, and adult E(2) treatment partially restored kisspeptin peptide expression. Finally, we showed that, after E2 treatment of WT and ArKO mice between either P5 and P15 or P15 and P25, AVPv/PeN kisspeptin peptide expression could be still masculinized at P5, but was feminized from P15 onward. In conclusion, the 2 kisspeptin neuronal populations (AVPv/PeN vs ARC) seem to be differentially organized and activated by E(2).