KNDy neurons are named for their co-expression of three neuropeptides, kisspeptin, neurokinin B (NKB) and dynorphin A (DynA). These cells, located in the hypothalamic arcuate nucleus (ARC), are associated with generation of gonadotropin-releasing hormone (GnRH)/luteinizing hormone (LH) pulses to control follicular growth and steroidogenesis. However, subcellular sorting mechanisms for secretory vesicles containing these neuropeptides have not been elucidated. In this study, we analyzed the localization pattern of kisspeptin, NKB and DynA in the ARC of the ovariectomized rat immediately treated with estrogen using immunoelectron microscopy. First, we identified neuropeptides by dual-labeled fluorescence immunohistochemistry, with results indicating all three neuropeptides co-express within individual ARC cells in female rats. Next, we investigated the subcellular localization pattern of kisspeptin, NKB, and/or DynA using post-embedding double immunoelectron microscopy, indicating that each type of neuropeptide is contained within separate and individual neurosecretory vesicles. This suggests sorting and packaging of kisspeptin, NKB and DynA is differentially regulated within KNDy neurons. Our findings facilitate understanding of regulatory mechanisms underlying kisspeptin secretion in KNDy neurons, and generation of GnRH/LH pulses induced by kisspeptin in the ARC. (C) 2015 Elsevier Ireland Ltd. All rights reserved.