Endothelial cells (EC) possess several protective thromboregulatory mechanisms that may be perturbed by cell activation or injury. Vascular ATP-diphosphohydrolase (ATPDase) has been demonstrated on both aortic EC and smooth muscle cells and may play a key regulatory role in hemostasis and platelet reactivity by converting extracellular ATP and ADP to AMP. We have examined the role of exogenous saturated or unsaturated fatty acids in the modulation of EC associated ATPDase activity in vitro. EC growth was not dramatically influenced by supplementation with fatty acids whereas viability was enhanced by oleic, butyrate and eicosapentaenoic acid. EC cultures supplemented with saturated or a monounsaturated (oleic acid) fatty acid(s) had markedly increased ATPDase activity, whereas those exposed to polyunsaturated fatty acids showed substantive decreases. Exogenous oleic acid could also protect against the significant loss of ATPDase activity, following exposure to reactive oxygen intermediates in vitro. We conclude that endothelial ATPDase activity may be regulated by exogenous fatty acids and that underlying mechanisms include alterations in the nature of the phospholipid composition of EC membranes that influence responses to oxidative stress reactions.