Despite refinements in treatment platforms such as incorporation of high-resolution imaging and real time motion tracking, the amount of radiation dose delivered safely to a particular tissue type is limited by both tissue intrinsic and extrinsic factors. FLASH radiotherapy (RT), delivery of relatively high radiation dose at dose rates several orders of magnitude higher than that of conventional (CONV) dose-rates, may change that paradigm due to normal tissue sparing characteristics of FLASH RT. Our Radiation Oncology Physics division developed a novel FLASH kV x-ray cabinet system for preclinical laboratory research. Here we utilize this technology to establish optimal radiation parameters for FLASH effect in brain tissue of a murine model. A range of total radiation dose and dose rates that are considered to be within the window for FLASH effect are being investigated. We have identified several measurable and clinically meaningful toxicity outcomes, including radiation necrosis and blood brain barrier integrity, to assess FLASH effects in brain tissue. A high-capacity rotating anode x-ray tube was implemented for FLASH irradiation, operating at 150 kVp with 0.025 mm Cu added filtration. A custom 3D printed immobilization tool was designed to reproducibly position animals for stereotactic irradiation of the brain. Dose and dose rate measurements were performed with calibrated radiographic film in kV solid water. Conventional dose rate irradiations were delivered using a standard laboratory irradiator (SARRP) operating at the same kVp and filtration. The depth-dose gradients in solid water were compared between the irradiators. Healthy 8-week-old C57BL6J mice were then irradiated with graded doses of FLASH and CONV x-rays (10- 60 Gy) using a single 1-cm wide AP field. Dose was prescribed to the center of the brain, at a depth of 3.25 mm. RT-induced necrosis is being monitored by serial biweekly MR imaging using a 7-T preclinical MRI machine. Histopathological changes will be assessed through H&E staining of harvested brain at 3-months post-irradiation (n = 5 per arm). FITC-Dextran infusion will be performed prior to necropsy to assess blood-brain barrier integrity. Animal setup for stereotactic FLASH brain irradiation was reproducible to within 0.5mm. Measured dose rates were 75.6 Gy/s for FLASH and 3.8 Gy/min for conventional irradiations. At 5-mm depth in solid water, the PDDs agreed to within 4% between SARRP and FLASH Irradiator. Biweekly T1 post and T2 weighted image acquisition is currently underway. Histology and FITC-Dextran data will be presented when available. We have successfully designed a high-precision platform to study x-ray FLASH effects in mice brain model. Evaluation of dose window for brain FLASH effect and molecular mechanisms of this phenomenon is an important step in the potential translation of FLASH RT for brain irradiation.