Several studies suggest that conjugated linoleic acid (CLA) isomers occuring naturally in milk and meat of ruminants influence atherosclerotic processes. The aim of this study was to investigate the effects of cis-9, trans-11 CLA und trans-10, cis-12 CLA on atherosclerosis-relevant parameters, vasoactive substances and pro-inflammatory parameters, in human arterial endothelial cells. To investigate the effect of both CLA isomers on the release of vasoactive substances playing an important role for maintenance of vascular homeostasis, human aortic endothelial cells (HAoEC) were incubated with 5 or 50 µmol/L of cis-9, trans-11 CLA or trans-10, cis-12 CLA for 24 h at 37 °C. After incubation period, the fatty acid composition in total lipids and phospholipids, formation of conjugated metabolites of CLA in total lipids, parameters of the eicosanoid synthesis and the nitric oxide synthesis as well as the release of endothelin-1 were determined. Incubation of HAoEC with the CLA isomers resulted in a significant incorporation of either CLA isomer and a significant isomer-specific formation of conjugated metabolites of CLA (CD) such as CD16:2 and CD20:2 fatty acids in a concentration dependent manner when compared with untreated endothelial cells. CLA isomers lowered the concentration of aracidonic acid in membrane phospholipids, activity of secretory phospholipase A2 as well as the release of eicosanoids (PGI2, TXA2, PGE2, PGF2α), nitric oxide and endothelin-1. But the ratio between the amounts of PGI2 and TXA2 released did not differ between control and cells treated with CLA isomers. To investigate the effect of both CLA isomers on pro-inflammatory parameters of the early phase of atherosclerosis, endothelial cells were incubated in medium without or with 5 or 50 µmol/L of either cis-9, trans-11 CLA, trans-10, cis-12 CLA or LA (as reference substance) for 20 h at 37 °C followed by a 4 h treatment with 2 ng/mL tumor necrosis factor (TNF)α. After incubation periods, the expression of adhesion molecules ICAM-1, VCAM-1 and E-selectin, the release of chemokines MCP-1 and IL-8 and the U937 monocyte adhesion to endothelial cells as well as the DNA-binding activity of the transcription factors NFκB and PPARγ were determined. Treatment of HAoEC with either CLA isomer or LA increased activity of PPARγ, but the TNFα-induced expression of adhesion molecules, release of chemokines, monocyte adhesion and activity of NFκB were almost not modulate. In conclusion, both CLA isomers cis-9, trans-11 CLA and trans-10, cis-12 CLA had no effects on the investigated atherosclerosis-relevant parameters in human arterial endothelial cells.