Orexin neuropeptides participate in the regulation of feeding as well as the regulation and maintenance of wakefulness and the cognitive functions. Orexin A and B share a common precursor, prepro-orexin and neurons are localized in the lateral hypothalamus. Physiological studies showed that these neurons are regulated by glutamatergic innervations. We aimed to assess the effects of kainic acid as a potent agonist for non-NMDA glutamate receptors in the activation of orexin neurons. We also analyzed the effect of glutamate antagonist CNQX, injected prior to kainic acid, on this activation. Expression of c-Fos protein was used as a marker for neuronal activation. Dual immunohistochemical labeling was performed for prepro-orexin and c-Fos and the percentages of c-Fos-expressing orexin neurons were obtained for control, kainic acid, and CNQX groups. Kainic acid injection caused statistically significant increase in the number of c-Fos-positive neurons when compared to control group (62.69 and 36.31%, respectively). Activation of orexin neurons was blocked, in part, by CNQX (43.36%). In the light of these results, it is concluded that glutamate takes part in the regulation of orexin neurons and partially exerts its effects through non-NMDA glutamate receptors and that orexin neurons express functional non-NMDA receptors.