Dietary free fatty acids have been reported to have various effects on the endothelium including the generation of nitric oxide. The goal of the current study was to determine the mechanism whereby free fatty acid causes an increase in nitric oxide synthesis. The specific hypothesis tested was that free fatty acid association with CD36, a class B scavenger receptor, induces the activation of endothelial nitric-oxide synthase (eNOS). A human microvascular endothelial cell line and a transfected Chinese hamster ovary cell system were used to determine which free fatty acids stimulate eNOS. Surprisingly, only myristic acid, and to a lesser extent palmitic acid, stimulated eNOS. The stimulation of eNOS was dose-and time-dependent. Competition experiments with other free fatty acids and with a CD36-blocking antibody demonstrated that the effects of myristic acid on eNOS required association with CD36. Further mechanistic studies demonstrated that the effects of myristic acid on eNOS function were not dependent on PI 3-kinase, Akt kinase, or calcium. Pharmacological studies and dominant negative constructs were used to demonstrate that myristic acid/CD36 stimulation of eNOS activity was dependent on the activation of AMP kinase. These data demonstrate an unexpected link among myristic acid, CD36, AMP kinase, and eNOS activity.Dietary fatty acids are involved in the development of numerous cardiovascular diseases; however, the reported molecular mechanisms and the physiological end points are often controversial. The controversy is in part because dietary fatty acids affect multiple systems (e.g. liver, lipoproteins, endothelium, macrophages) and often have synergistic and/or antagonistic influences on the development of disease and because different fatty acids have different effects (1-4). In the present study we focused on the effects of free fatty acids on endothelial nitric-oxide synthase (eNOS) 1 activity. Few studies have examined the link between free fatty acids and eNOS activity. However, numerous studies have demonstrated that acylation of eNOS with myristic acid and palmitic acid can alter the subcellular localization of eNOS with subsequent profound effects on eNOS activity (5-8). A recent study by Esenabhalu et al. (9) showed that overloading endothelial cells with oleic acid caused an attenuation of calcium signaling, which resulted in a decrease in nitric oxide production. However, Lynn et al. (10) demonstrated that lower concentrations of oleic acid did not affect nitric oxide production. Interestingly, Lynn et al. also demonstrated that eicosapentaenoic acid did increase insulinstimulated nitric oxide production (10). These findings suggest that both the concentration and type of fatty acid can influence the activity state of eNOS and the subsequent production of nitric oxide. The mechanism(s) of how extracellular free fatty acid influences eNOS activity is not understood. One possibility is through interaction with CD36, a class B scavenger receptor that is expressed on the surface of numerous ce...