Ectoenzymes expressed on the surface of vascular cells and leukocytes modulate the ambient nucleotide milieu. CD73 is an ecto-5' nucleotidase that catalyzes the terminal phosphohydrolysis of AMP and resides in the brain on glial cells, cells of the choroid plexus, and leukocytes. Though CD73 tightens epithelial barriers, its role in the ischemic brain remains undefined. When subjected to photothrombotic arterial occlusion, CD73(-/-) mice exhibited significantly larger (49%) cerebral infarct volumes than wild-type mice, with concordant increases in local accumulation of leukocyte subsets (neutrophils, T lymphocytes, macrophages, and microglia). CD73(-/-) mice were rescued from ischemic neurologic injury by soluble 5'-nucleotidase. In situ, CD73(-/-) macrophages upregulated expression of costimulatory molecules far more than wild-type macrophages, with a sharp increase of the CD80/CD86 ratio. To define the CD73-bearing cells responsible for ischemic cerebroprotection, mice were subjected to irradiative myeloablation, marrow reconstitution, and then stroke following engraftment. Chimeric mice lacking CD73 in tissue had larger cerebral infarct volumes and more tissue leukosequestration than did mice lacking CD73 on circulating cells. These data show a cardinal role for CD73 in suppressing ischemic tissue leukosequestration. This underscores a critical role for CD73 as a modulator of brain inflammation and immune function.