Ecto 5´-nucleotidase (CD73) is a key enzyme responsible for formation of cardioprotective adenosine from extracellular nucleotides as well as an important GPI-anchored surface molecule involved in cellular signalling. Lysophosphatidylcholine (LPC) and platelet activating factor (PAF) formed during inflammation and ischemia are potent lipid mediators that enhance the extracellular adenosine production in HUVEC. This calcium and PKC independent effect is concentration and time dependent. Increased production of adenosine is in particular found in the supernatant of stimulated cells. To determine the yet unclear mechanism of increased AMP hydrolysis by phospholipids we measured the extracellular dephosphorylation rate of ethenoAMP to ethenoadenosine of HUVEC by HPLC using LPC and PAF as stimuli. To test for the specific importance of CD73 in the mediation of the LPC and PAF responses we generated CD73 negative HUVEC by using a GPI cleaving enzyme that cleaves CD73 from the membrane. CD73 negative HUVEC revealed a significantly reduced conversion of ethenoAMP to ethenoadenosine. Interestingly, we observed that the cleaved enzyme in the supernatant was more active than the membrane bound form. Because PAF synthesis is mediated via a remodelling pathway in which membrane phospholipids are converted by a phospholipase A2 into metabolic inactive lyso-PAF, which is then acetylated by acetyl CoA:lyso-PAF acetyltransferase to form metabolic active PAF, we also investigated if the inactive metabolite lyso-PAF can enhance CD73 activity. These experiments showed that also lyso-PAF increased the production of adenosine via CD73 in a concentration dependent manner. In conclusion, CD 73 mediates the enhanced conversion of extracellular AMP to adenosine following exposure to phospholipids. In part, this effect may be brought about by cleavage of the GPI-anchor permitting diffusion supported interaction of both, substrate and enzyme, which may enhance catalytic effectiveness.