Background and Purpose-We, for the first time, performed in vivo x-ray angiography in the mouse brain using SPring-8, a third-generation synchrotron radiation facility. Methods-A thin PE-50 tube was placed in the unilateral external carotid artery in adult male C57BL/6J mice. While maintaining the blood flow in the internal carotid artery, 33 L of contrast agent was injected and then selective angiography of the hemisphere was performed. Results-The average diameters of cerebral artery were as follows: 142.5Ϯ7.90 m in middle cerebral artery, 138.3Ϯ9.35 m in anterior cerebral artery, 120.5Ϯ5.53 m in posterior cerebral artery, and 162.6Ϯ10.87 m in internal carotid artery (nϭ5). To demonstrate the changes in diameter, we induced hypercapnia and detected the dilatation of the vessels between 121% and 124% of the original diameters (nϭ5). We also repeated angiography in the mice before and after intracarotid injection of vasodilatation drugs papaverine hydrochloride, ATP disodium, and fasudil hydrochloride hydrate and demonstrated the chronological changes in the diameters in each artery at 1, 5, 15, and 30 minutes after injection (nϭ1 for each drug). Conclusions-Using only a minimum volume of the contrast agent, synchrotron radiation enables us to study x-ray angiography in the mouse brain. The morphology of the vessels can be clearly observed under physiological conditions. The diameters and their changes can also be successfully studied in vivo. (Stroke. 2006;37:1856-1861.)