Reduced nitric oxide (NO) availability is associated with the development of atherosclerosis. Upregulation of endothelial nitric oxide synthase (eNOS) activity is pursued as a strategy for the prevention of cardiovascular diseases. The polyphenol hydroxytyrosol (HT) which is present in olive oil and red wine, is regarded to be partly responsible for the beneficial effects associated with olive oil consumption and has shown antiatherogenic activity in vitro and in vivo. To elucidate the underlying molecular mechanisms, we investigated possible effects of HT on the endothelial nitric oxide synthase (eNOS). We used human endothelial cells (EA.hy926) and examined eNOS on three different levels, addressing eNOS promoter transactivation, eNOS enzyme activity and nitric oxide availability. Cells were treated with a broad range of HT concentrations (from 10 nM to 100 microM) and for different incubation times (15 min to 24 h). HT did not exert significant positive effects on eNOS in any of our assay systems. Neither did we find evidence for a possible synergism between the red wine polyphenol resveratrol and HT. We conclude that a direct modulation of eNOS is unlikely to account for the antiatherogenic properties of HT under non-inflammatory conditions.