Body weight regulation depends on neuronal signaling by adiposity-related hormones such as insulin and leptin. Activation of receptors for these hormones induces cell signaling via the insulin receptor substrate/phosphatidylinositol 3-kinase (IRS-PI3K) pathway, and growing evidence from knockout models implicates IRS-2 as a key component of this signal transduction mechanism. As a first step towards the identification of brain areas that utilize IRS-PI3K signaling in the control of energy homeostasis, we used immunohistochemical techniques to investigate the neuronal distribution of IRS-2 protein in rat brain. In the hypothalamus, strong IRS-2 staining was detected chiefly in the arcuate (ARC), ventromedial (VMN) nucleus and parvocellular paraventricular nucleus (PVN). Within the ARC, IRS-2 was co-localized with alpha melanocyte stimulating hormone (alpha-MSH) as well as neuropeptide Y (NPY). In the hindbrain, IRS-2 staining was detected in the area postrema (AP), medial nucleus of the solitary tract (mNTS), dorsal motor nucleus of the vagus nerve (DMV) and the hypoglossal nucleus (HN). Co-localization studies in the mNTS demonstrated the presence of IRS-2 in catecholamine neurons. IRS-2 protein was also found in the ventral tegmental area (VTA), an important area for reward perception, and was detected in dopamine neurons in this brain area. In summary, neurons containing IRS-2 immunoreactivity were identified in forebrain, midbrain and hindbrain areas and in cell types that are crucial for the control of food intake and autonomic function. An improved understanding of mechanisms underlying normal and abnormal energy homeostasis may be gained by analysis of the role played by signaling through IRS-2 in these brain areas.