Inflammation of endothelium is closely associated with the development of atherosclerosis. Extracellular adenosine levels are increased at the sites of inflammation. Such adenosine increase may serve to protect tissues from injury and potentially slow down the progression of atherosclerosis because adenosine is known to possess anti-inflammatory property. However, until now, the mechanism leading to the increased extracellular adenosine level during inflammation has not been understood. Two proteins are suspected to play roles in this mechanism: 1) equilibrative nucleoside transporters (ENTs), which control the rate of adenosine transport across cells; 2) ecto-5’nucleotidase (ecto-5’Nu), which metabolizes extracellular adenosine monophosphate into adenosine. In our study, lipopolysaccharide (LPS) was used to induce inflammation in human umbilical vein endothelial cells (HUVEC). The results from RT-PCR and [3H]adenosine transport assay revealed that the expressions and activities of ENTs in HUVEC were not altered by LPS stimulation. In contrast, biochemical assay demonstrated that the ecto-5’Nu activity in HUVEC was increased after incubation with LPS for 24 hours. This effect was dose-dependent, with an IC50 value of 1 ng/ml. In addition, western blot analysis showed that the protein expression of ecto-5’Nu in HUVEC was higher after stimulation by LPS. LY294-002, a phosphatidylinositol 3-kinase (PI3K) inhibitor, was found to reduce the LPS-stimulated ecto-5’Nu activity. Therefore, it is suggested that the increase in extracellular adenosine level during inflammation may be, at least in part, related to the up-regulation of ecto-5’Nu through a PI3K signaling pathway.