The objective of this study was to determine the effects of cis-9, trans-11 and trans-10, cis-12 CLA on the release of vasoactive eicosanoids and nitric oxide (NO) in human aortic endothelial cells. Experiments were conducted in which cells were incubated with these fatty acids, and the concentrations of various eicosanoids [6-keto prostaglandin (PG) F-1alpha as a stable product of PGI(2), thromboxane (TX) B-2 as a stable product of TXA(2), and PGE(2)] and NO in the medium were determined. Cells treated with 50 mumol/L of either cis-9, trans-11 or trans-10, cis-12 CLA released less of all of the eicosanoids and NO than control cells treated with medium alone (P < 0.05). The ratio between the amounts of 6-keto-PGF(1alpha) and that of TXB2 released did not differ between control cells and cells treated with either CLA isomer. Moreover, cells treated with 50 mumol/L of cis-9, trans-11 or trans-10, cis-12 CLA had a lower amount of arachidonic acid in their phosohatidylethanolamine fraction and a lower mRNA concentration and activity of secretory phospholipase A(2) than control cells (P < 0.05). These data suggest that eicosanoid formation was impaired by a reduced availability of arachidonic acid for the cyclooxygenase pathway. In conclusion, this study shows that cis-9, trans 11-CLA and trans-10, cis-12 CLA influence the release of various eicosanoids and NO from human aortic endothelial cells. The effects observed in this study might be important because eicosanoids and NO released from endothelial cells are involved in the regulation of vessel tone and platelet aggregation. The results of the present study suggest that both CLA isomers had unfavorable effects on endothelial function. J. Nutr. 133: 4083-4089, 2003.