Adenosine is thought to prevent or reduce the increase in permeability, which is a hallmark of oxidant injury to endothelium. However, the effect of adenosine on endothelial cells directly exposed to oxidant species has not been demonstrated in vitro. By measuring the passage of Evan's blue dye-labeled albumin across confluent monolayers, we demonstrated the ability of adenosine (0.1-100 µM) to lower basal permeability of human umbilical vein endothelial cells in a concentration-dependent fashion and prevent the permeability increase induced by exposure of the cells to xanthine plus xanthine oxidase (X/XO). Whereas pretreatment of monolayers for 10 min with adenosine (10 and 100 µM) prevented the X/XO-induced permeability increase, these same concentrations of adenosine failed to increase intracellular adenosine 3',5'-cyclic monophosphate in X/XO-exposed cells. The protective effect of adenosine on endothelial monolayers was mimicked by adenosine amine congener and 5'-(N-ethylcarboxamido)adenosine but not by other agonists examined. Hence, the protective effect of adenosine against oxidant injury may include an adenosine 3',5'-cyclic monophosphate-independent mechanism by direct action of adenosine at A₁ receptors on endothelial cells.