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BACKGROUND AND AIMS: Recent anatomical and transgenic studies in mice provide evidence for the critical involvement of neuronal nitric oxide synthase (nNOS) neurons in metabolic- and sex steroid regulation of fertility. In humans, the nature and sites of putative interactions between nNOS neurons and the hypothalamic circuitry controlling reproduction is entirely unknown. METHODS: To study anatomical sites where such interactions may occur, we have carried out immunohistochemical experiments on formalin-fixed post mortem histological samples of postmenopausal women. RESULTS: The perikarya and fibers of nNOS-immunoreactive neurons were widely distributed in preoptic/hypothalamic tissue sections of the human. High labeling intensities were observed in the diagonal band of Broca, the medial preoptic area and the hypothalamic ventromedial, dorsomedial, infundibular (Inf), paraventricular and supraoptic nuclei. At many of these sites including the Inf, gonadotropin-releasing hormone (GnRH) neurons were often surrounded by nNOS-immunoreactive neurons. In addition, nNOS cells of the Inf received frequent axo-somatic and axo-dendritic neuronal contacts from the local kisspeptin neurons. Unlike in the mouse arcuate nucleus where nNOS and kisspeptin neurons are distinct, these two cell populations showed a partial overlap in the human; 1–20% of kisspeptin-immunoreactive cell bodies in three different individuals (12.98±3.7%) showed co-labeling for nNOS. The abundant kisspeptin input to nNOS cells, together with the close anatomical relationship between nNOS and GnRH neurons, raise the possibility that kisspeptin neurons act on nNOS cells to influence GnRH neurons indirectly, in addition to activate GnRH neurons directly. Evidence for such indirect communication route has been reported recently in mice (Hanchate et al. 2012, J Neurosci 32: 932–945).