Since the peripheral serotoninergic pathway is involved in the development of radiation-induced nausea and vomiting, referred to as radiation sickness, serotonin 5-HT3 receptor antagonists are used as a preventive measure, although patients still suffer from these symptoms. Glutamate is known as the excitatory neurotransmitter and is involved in various autonomic symptoms. We investigated the effect of radiation on glutamate release in rats, as measured by in vivo brain microdialysis, and the effects of glutamate receptor antagonists on radiation-induced pica, which can be used as a behavioral assessment of radiation sickness in rats. A microdialysis probe was inserted into the hypothalamus of rats that received 4 Gy total-body irradiation (TBI) with or without pretreatment of 5-HT3 receptor antagonist (granisetron, 0.1 mg/kg, i.p.), and dialysates were collected for 3 h after TBI and subjected to HPLC assay of glutamate. In addition, rats were intracerebroventricularly injected with NMDA receptor antagonist (MK-801: 3 μg/rat) or AMPA receptor antagonist (CNQX: 1 μg/rat) before TBI, and radiation-induced pica was determined. An increase in glutamate release was observed within 1 h postirradiation. The increased glutamate release was suppressed by granisetron. We also found that CNQX, but not MK-801, effectively inhibited radiation-induced pica. These results indicate that the hypothalamic glutamatergic system contributes to radiation-induced pica through the AMPA receptors.