Overlapped in the tuberal hypothalamic area (THA), melanin-concentrating hormone (MCH) and hypocretin (Hcrt) neurons contribute to the integrated regulation of food intake, energy regulation and sleep. Recently, physiological role in appetite suppression has been defined for a novel hypothalamic molecule, nesfatin-1. Acute i.c.v. infusion of nesfatin-1 (nesf-1) promotes anorexia whereas chronic treatment reduces body weight in rats. This satiety molecule is expressed in neurons from areas prominently involved in appetite regulation including THA. We therefore sought functionally relevant to determine whether nesf-1 might be a reliable signaling marker for a new cell contingent within THA, in addition to MCH and Hcrt neurons. Thus, we completed a detailed topographical mapping of neurons immunostained for nesf-1 (nesf-1+) together with cell quantification in each discrete nucleus from THA in the rat. We further combined the immunodetection of nesf-1 with that of MCH or Hcrt to assess possible co-expression. More than three quarters of the nesf-1+ neurons were encountered in nuclei from the lateral half of THA. By double immunofluorescent staining, we showed that all neurons immunoreactive for melanin concentrating hormone (MCH+) neurons depicted nesf-1 immunoreactivity and approximately 80% of the nesf-1+ neurons were labeled for MCH. Maximal co-expression rates were observed in the lateral THA containing approximately 86% of the double-labeled neurons plotted in THA. The present data suggest that nesf-1 co-expressed in MCH neurons may play a complex role not only in food intake regulation but also in other essential integrative brain functions involving MCH signaling, ranging from autonomic regulation, stress, mood, cognition to sleep.