After an inflammatory stimulus, lymphocyte migration into draining lymph nodes increases dramatically to facilitate the encounter of naive T cells with Ag-loaded dendritic cells. In this study, we show that CD73 (ecto-5′-nucleotidase) plays an important role in regulating this process. CD73 produces adenosine from AMP and is expressed on high endothelial venules (HEV) and subsets of lymphocytes. Cd73−/− mice have normal sized lymphoid organs in the steady state, but∼ 1.5-fold larger draining lymph nodes and 2.5-fold increased rates of L-selectin-dependent lymphocyte migration from the blood through HEV compared with wild-type mice 24 h after LPS administration. Migration rates of cd73+/+ and cd73−/− lymphocytes into lymph nodes of wild-type mice are equal, suggesting that it is CD73 on HEV that regulates lymphocyte migration into draining lymph nodes. The A 2B receptor is a likely target of CD73-generated adenosine, because it is the only adenosine receptor expressed on the HEV-like cell line KOP2. 16 and it is up-regulated by TNF-α. Furthermore, increased lymphocyte migration into draining lymph nodes of cd73−/− mice is largely normalized by pretreatment with the selective A 2B receptor agonist BAY 60-6583. Adenosine receptor signaling to restrict lymphocyte migration across HEV may be an important mechanism to control the magnitude of an inflammatory response.