Introduction: TNF-α is a pro-inflammatory cytokine and harmful circulating stimulus (1). Endothelial cells are targets of harmful TNF-α (1) and high concentrations of TNF-α can set off signalling events that can lead to endothelial injury. Oleanolic Acid (OA) is a triterpenoid found in food and plants (2) and has previously been shown to have antioxidant properties (3). Combination treatment with TNF-α and OA has not been investigated before. Aims: Investigating the effects of TNF-α (20ng/ml, 24h), OA (40µM, 24h) or OA pre-treatment+TNF-α on the eNOS-NO biosynthesis pathway, oxidative stress and cell viability in vitro. Methods: CMECs (Rattus norvegicus) were incubated with: TNF-α and/or OA-containing medium. At the end of incubation, samples were prepared for flow cytometric (FC) and western blot analyses (WB). Intracellular levels of nitric oxide (NO) and reactive oxygen species (ROS) were determined by FC analyses of DAF-2/DA and DHR-123 fluorescence. eNOS, iNOS, PKB, caveolin-1, HSP90, nitrotyrosine, p22-phox, cleaved caspase-3, cleaved PARP and Iκβα were determined by WB measurements. Results: TNF-α resulted in decreased NO levels (77.32±0.87 vs. 100; p<0.05). TNF-α and TNF-α+OA resulted in reduced phospho/total ratio eNOS (0.79±0.05; 0.79±0.04 vs. 1; p<0.05). A reduction in caveolin-1, HSP90 and phospho/total ratio PKB was also seen in TNF-α and TNF+OA (p<0.05). Despite reduced ROS levels observed with TNF-α, p22-phox expression increased in all three groups (1.93±0.05; 1.38±0.09; 2.06±0.04 vs. 1; p<0.05). Reduced DHR-123-sensitive ROS corresponded with reduced nitrotyrosine in TNF-α and OA groups (p<0.05). Caspase-3 increased in TNF-α (1.12±0.02 vs. 1; p<0.05), whereas OA resulted in reduced caspase-3 (0.94±0.01 vs. 1; p<0.05). No changes were observed in cleaved PARP. Reduced Iκβα expression was observed in all treatments (p<0.05). Discussion: The TNF-α-induced decrease in NO seemed to be associated with a decrease in phospho/total eNOS ratios, accompanied by reduced PKB ratio, HSP90 and caveolin-1. The TNF-α-induced decrease in ROS levels, contradicted the increase observed in p22-phox expression. The reduced DHR-123-sensitive ROS levels corresponded with a reduction in nitrotyrosine. TNF-α seemed to be pro-apoptotic, whereas OA was anti-apoptotic. The increase in p22-phox (TNF-α, OA and TNF+OA) did not translate in increased ROS generation. In conclusion, TNF-α induced an overall adaptive response with early signs of increased NADPH-oxidase activity and apoptosis. OA seemed to be protective, though it was not able to reverse the effects seen with TNF-α.