Transient hypoxia stimulates endothelial cell (EC) proliferation by a mechanism involving activation of the MEK/ERK pathway and NADPH oxidase. Here we studied whether this mechanism triggers tube formation and whether p38MAPK is a signalling element involved in this pro-angiogenic effect. EC were exposed to hypoxia (Po2 < 10 mmHg) or normoxia (Po2 = 140 mmHg) for 2h, followed by 24h of reoxygenation. Transient hypoxia activated ERK1/2, NADPH oxidase and p38MAPKa (p38a) (immunoprecipitation, western blot). After reoxygenation cell number was increased by 65% compared to normoxic control (P<0.05,n=5) and EC formed tube-like structures in a 2D sprouting assay (collagen gel). Inhibition of MEK1/2 by PD-98059 or NADPH oxidase by antisense oligonucleotides against p22phox reduced activation of p38a indicating that MEK\ERK and NADPH oxidase are upstream of p38a. These maneuvers also abolished the hypoxia-induced activation of cell cycle (pRb- phosphorylation, BrdU incorporation), cell number, and tube formation. Inhibition of p38a (SB-202190, p38a-specific inhibitor, or SiRNA) reduced hypoxia-induced proliferation and completely abolished tube formation. Conclusion: p38&alpha is a central pro-angiogenic signalling element downstream of MEK/ERK and NADPH oxidase induced by transient hypoxia. It does not only trigger cell proliferation but also stimulate formation of tube-like structures.