Dietary fatty acids (FAs) are known to modulate endothelial dysfunction, which is the first stage of atherosclerosis. However, their exact role in this initial phase is still unclear. The effects of isolated or combined (by 2) purified FAs from the main FA families were studied on the vascular response of isolated thoracic aorta in healthy rats to get a better understanding of the mechanisms of action of dietary FAs in regulating vascular endothelial function. Cumulative contraction curves to phenylephrine and relaxation curves to carbachol and then to sodium nitroprusside were obtained in the absence or presence of the FAs studied allowing endothelium-dependent and endothelium-independent ability of the smooth muscle to relax to be assessed in each experimental group. The endothelium-dependent vasodilator response to carbachol was lowered by eicosapentaenoic acid, whereas it was not altered either by docosahexaenoic acid alone or by combined eicosapentaenoic acid-docosahexaenoic acid, oleic acid, or stearic acid, and it was increased by linoleic acid (LA). A decreased phenylephrine-induced contraction was observed after incubation with arachidonic acid and with stearic acid. On the other hand, the endothelium-dependent relaxation was reduced by the addition of combined LA-arachidonic acid and LA-oleic acid. In conclusion, these data point out the differential effects of different types of FAs and of FAs alone vs combined on vascular reactivity. The complex nature of these effects could be partially linked to metabolic specificities of endothelial cells and to interactions between some FAs.