Long term use of sugars can induce excess caloric intake and/or obesity. To evaluate the effects of sugar intake on different regions of the hypothalamus (the brain's control center for energy homeostasis) we first developed and then evaluated a microscope-assisted dissection method. Because of the small size of the paraventricular nucleus, we validated the samples by measuring several hormones mainly synthesized in the paraventricular nucleus. These include corticotropin-releasing hormone, oxytocin, arginine vasopressin and thyrotropin releasing hormone. We measured the mRNA expression of each of these hormones using quantitative PCR and detected them principally in the paraventricular nucleus. We further evaluated the effects of various sugar solutions on the expression of several important hypothalamic neuropeptides because they play a pivotal role in energy homeostasis. We provided Sprague Dawley rats 24 hour access to 15% solutions of glucose, fructose, sucrose or high fructose corn syrup. We then measured the expression of several neuropeptides in different hypothalamic regions, all of which were previously shown to be influenced by sugar consumption (mainly based on the results from a series of PCR arrays). Additionally, we measured plasma leptin, known for its close correlation with body fat mass. As expected, rats that had access to sugar solutions consumed less chow. However, rats with free access to sugar solutions maintained a similar amount of energy intake compared with control. Of the four sugars tested, only fructose decreased expression of cholecystokinin significantly, whereas glucose and sucrose significantly increased the expression of tumor necrosis α only in the paraventricular nucleus, not in the ventromedial nucleus or the lateral hypothalamic area. Fructose and sucrose decreased growth hormone expression in the ventromedial nucleus. Glucose increased dopamine receptor D1A expression in the paraventricular nucleus only. We conclude that 24 hour free access to different sugars can influence the expression of several hypothalamic neuropeptides in different ways and these changes are region dependent. Changes in the expression of these neuropeptides do not disrupt the total energy intake immediately but may contribute to the obesity caused by long term intake of sugars.