Formation of ATP from ADP at the external surface of vascular endothelial cells has been attributed to plasma membrane ATP synthase (PM-ATP synthase), ecto-adenylate kinase (ecto-Ak) and/or ecto-nucleoside diphosphokinase (ecto-NDPK). These enzymes or their catalytic products have been causatively linked to the elaboration of vascular networks and the regulation of capillary function. The amount of ATP generated extracellulary is small, requiring sensitive analytical methods for quantification. Human umbilical vein endothelial cells (HUVECs) were used to re-visit extracellular ATP synthesis using a reliable tetrazolium reduction assay and multi-well plate cultures. Test conditions compatible with Ak stability were established. Extracellular Ak activity was found to be less than 1% of the total (intracellular + extracellular), raising the possibility that the external enzyme could have leaked from living cells and/or a few dying cells. To determine whether Ak was inadvertently leaked from the cells, the activity of another cytoplasmic enzyme, glucose-6-phosphate dehydrogenase (GPdh), was also measured. GPdh is present in the cytoplasm in similar abundance to Ak. The GPdh activity ratio (extracellular:total) was found to be similar to that for Ak. Since GPdh in the medium was probably due to leakage, other cytoplasmic macromolecules, including Ak, should be released proportionately from the cells. The role of PM-ATP synthase in extracellular ATP formation was examined using Hanks’ solution +/- selective inhibitors of Ak and ATP synthase activities. With Ap5A (inhibitor of Ak), no extracellular ATP synthesis was detected while with oligomycin, piceatannol and aurovertin (inhibitors of F1F0-ATP synthase or F1-ATPase activities) no inhibition of extracellular ATP synthesis was observed. Ak activity, alone, can account for the observed extracellular ATP synthesis. The possible impact of ADP impurity in the assays is discussed.