In clinical studies, omega-3 fatty acids (w-3 FA) have shown to improve the outcome of critically ill patients. Ecto-5'-nucleotidase (CD73), a GPI anchored protein located in lipid rafts, is the key enzyme that leads to the formation of extracellular adenosine which can activate cell protective signalling pathways. Previous studies suggested that modulation of the lipid bilayer organization by the omega-3 fatty acid docosahexaenoic acid (DHA) can increase the enzymatic activity of CD73. As the pulmonary system seems to be a major site of beneficial actions of omega-3 FA, our project aimed at defining the effect of DHA (25 to 100 mM) on CD73 protein content and enzymatic activity in human pulmonary microvascular endothelial cells (HPMEC) in comparison with human umbilical vein endothelial cells (HUVEC). Total CD73 protein content was determined by western blotting and plasma membrane abundance was quantified by flow cytometry. Enzymatic activity was determined by conversion of the exogenous substrate etheno-AMP (eAMP) to etheno-adenosine (eAdo) and quantified by HPLC. Furthermore, ATP-release of DHA-treated cells and the effects of the unhydrolyzable ATP-analogue ATP-gS on eAMP-conversion were studied. At 48 h after DHA treatment, we observed significant dose-dependent increases in CD73 plasma membrane abundance (HPMEC +28%, p=0.006; n=8; HUVEC +22% p=0.02, n=6) and total protein content (HPMEC +26% p=0.043, n=5; HUVEC 30% n=2). This coincided with a 25% increase in eAMP-conversion (p=0.002, n=5) in cell membrane preparations. In contrast, eAMP conversion was decreased by 15 to 20% in both HPMEC (p<0.001, n=6) and HUVEC (P=0.039, n=5) when intact cells were studied. In parallel with the decreased CD73 activity, ATP release was about 3-fold elevated in HPMEC. In further experiments, we found that the unhydrolyzable ATP-analogue ATP-gS dose-dependently inhibited CD73 activity. These results demonstrate that DHA can increase CD73 at the protein level. This regulatory effect is similar in microvascular lung endothelial cells and in HUVECs. While in cell membrane fractions the enzymatic activity determined as conversion of eAMP to eAdo correlates with cellular CD73 protein content, in intact cells the situation seems to be more complex: here, eAMP conversion is decreased after DHA treatment while on the other side, ATP-release by DHA-treated endothelial cells is enhanced. As the unhydolyzable ATP-analogue ATP-gS also inhibits eAMP-conversion in our model, this enhanced ATP release account for the attenuation of eAMP conversion observed.