The protein fragment nesfatin-1 was recently implicated in the control of food intake. Central administration of this fragment results in anorexia and reduced body weight gain, whereas antisense or immunological nesfatin-1 antagonism causes increased food intake and overweight. Nesfatin-1 is derived from the precursor nucleobindin-2 (NUCB2). To identify the neurocircuitry underpinning the catabolic effects of NUCB2/nesfatin-1, we have used in situ hybridization and immunohistochemistry to map the distribution of this protein and its mRNA in the rat CNS and performed double-labeling experiments to localize its expression to functionally defined neuronal populations. These experiments confirm previous observations but also present several novel NUCB2 cell populations. Both NUCB2 mRNA and nesfatin-like immunoreactivity was most concentrated in the hypothalamus, in the supraoptic, paraventricular, periventricular and arcuate nuclei and the lateral hypothalamic area/perifornical region. Additionally, outside of the hypothalamus, labeling was observed in the thalamic parafascicular nucleus, the Edinger-Westphal nucleus, locus coeruleus, ventral raphe system, nucleus of solitary tract and in the preganglionic sympathetic intermediolateral cell column of the spinal cord, and the pituitary anterior and intermediate lobes. In neurons, immunoreactivity was almost exclusively confined to perikarya and primary dendrites with virtually no labeling of axonal terminals. Double-labeling immunohistochemistry revealed colocalization of nesfatin with vasopressin and oxytocin in magnocellular neuroendocrine neurons, thyrotropin-releasing hormone, corticotropin-releasing hormone, somatostatin, neurotensin, and growth-hormone-releasing hormone in parvocellular neuroendocrine neurons, pro-opiomelanocortin (but not neuropeptide Y) in the arcuate nucleus and melanin-concentrating hormone (but not hypocretin) in the lateral hypothalamus. Furthermore, nesfatin was extensively colocalized with cocaine- and amphetamine-regulated transcript in almost all NUCB2-expressing brain regions. These data reveal a wider distribution of NUCB2/nesfatin-1 than previously known, suggesting that the metabolic actions of this protein may involve not only feeding behavior but also endocrine and autonomic effects on energy expenditure. In addition, the subcellular distribution of nesfatin-like immunoreactivity indicates that this protein may not be processed like a conventional secreted neuromodulator.