Transient hypoxia can prevent apoptotic death in human endothelial cells from different origins. Here the molecular mechanism of this protective effect is analysed. Apoptosis was induced in HUVEC by either serum deprivation or TNFa (20 ng/ml) for 24 hours. Afterwards cells were exposed to hypoxia (2h, Po2<10 mmHg) followed by reoxygenation for 24 hours (Po2=140 mmHg). Serum deprivation reduced cell number to 47%. Within this population 52% of the cells were apoptotic (Annexin V staining, FACS). In contrast, transient hypoxia reduced cell number to only 74%, and decreased apoptosis to 26% of the surviving cell population. Transient hypoxia had similar effects on TNFa-induced apoptosis. Hypoxia induced activation of ERK1/2 (Western blot). Inhibition of ERK 1/2 by antisense oligonucleotides against ERK 1/2 abolished the effect of transient hypoxia on apoptotic cell death. Transient hypoxia induced phosphorylation of the pro-apoptotic Bad at serine 112 (Western blot), a specific phosphorylation site of ERK 1/2 leading to its inactivation. Moreover, it reduced cytochrome C release from mitochondria (cell fractionation) and caspase 3 activation (Western blot) induced by serum deprivation. Inhibition of ERK 1/2 abolished the effect of hypoxia on all three parameters. Conclusion: Transient hypoxia protects endothelial cells from apoptotic execution by an ERK 1/2-dependent signalling mechanism targeting Bad, Cytochrome C and Caspase 3.