1.Melanin-concentrating hormone (MCH) and orexin-containing neurons participate in hypothalamic circuits that control energy homeostasis. While these two systems have projections to widespread target areas within the central nervous system, little is known about intrinsic characteristics and the molecular composition of both the MCH and orexin neurons themselves. 2. By a combinatory approach of quantitative immunocytochemical identification and analysis with laser microdissection and semi-quantitative Real-time RT-PCR, here we present multi-transcriptional profiling of MCH and orexin neurons in the rat lateral hypothalamus. 3. Immunocytochemical analysis showed that orexin peptide expression was increased after fasting both during the activity and resting period of rats, whereas MCH peptide content was only clearly upregulated at resting phase. Subsequent transcriptional profiling showed distinct expression patterns of MCH, orexin and cocaine-amphetamine regulated transcript (CART) between MCH and orexin neurons. A low expression level of dynorphin was found both in MCH and orexin neurons. Receptor expression profiles, reflecting interaction with neuropeptide Y, melanocortins, leptin, glucocorticoids and GABA, showed approximately similar expression patterns among the MCH and orexin neuronal systems. Expression of glutamate- and GABA-markers revealed a possible contributory role of both glutamate and GABA in functional output of MCH and orexin neurons. 4. This method allowed differential screening at mRNA level after immunocytochemical neuron identification and analysis in heterogeneous brain regions, which can further specify functioning of the individual neurons. With respect to MCH and orexin neurons, this study emphasizes that these neurons are targets for stimulatory and inhibitory signals from other brain regions including the arcuate nucleus and the general circulation. Additionally, both glutamate and GABA appear to be involved in MCH and orexin neuronal functioning related to feeding and regulation of the energy balance.