To study the ability of long-chain trans fatty acids (FA) to be incorporated and metabolized into endothelial cells, bovine aortic endothelial cells were incubated with medium enriched eicosapentaenoic acid (EPA) bound to albumin (M2) or one of its geometrical isomers: 20:5 5c,8c,11t,14c,17c (M3), 20:5 5c,8c,11c,14c,17t (M4), or 20:5 5c,8c,11t,14c,17t (M5). After 48 h of incubation, supernatant and cells were harvested and their lipids were analyzed, including prostacyclin synthesis. EPA and 22:5n-3 of endothelial cells incubated with M2 were, respectively, three and two times higher than in control cells (incubated in M1, without any fatty acid added), whereas 22:6n-3 increased only in the supernatant, suggesting its release after biosynthesis. However, 18:2n-6 and 22:4n-6 decreased (about 30%). Trans 20:5 isomers represented 4.7, 3.9, and 5.2% of total phospholipid FA in endothelial cells incubated with M3, M4, and M5, respectively. They were elongated into trans 22:5 and trans 24:5, as revealed by gas chromatography-mass spectrometry and gas chromatography-Fourier transform infrared analysis. In cells incubated with M2, M3, M4, and M5, prostacyclin synthesis was inhibited by 49.0, 62.5, 60.5, and 72.0%, respectively. This effect may be due to less available arachidonic acid in the cells and to a competition between EPA isomers and AA at the level of cyclooxygenase pathway, as it was demonstrated that 20:5 delta17t was metabolized by this enzyme.